2β-Chloromethyl-2α-methylpenam-3α-carboxylic acid sulfone methylene diol mixed esters

ABSTRACT

2β-Chloromethyl-2α-methylpenam-3α-carboxylic acid sulfone and salts and esters thereof were synthesized and found to be potent inhibitors of β-lactamases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of co-pending application Ser. No.271,744, filed June 8, 1981, now U.S. Pat. No. 4,380,512 patented Apr.19, 1983 which is a continuation-in-part of my prior, application Ser.No. 214,831 filed Dec. 11, 1980 and now abandoned which was in turn acontinuation-in-part of my prior, application Ser. No. 113,894 filedJan. 21, 1980 and now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The novel chemicals of the present invention include potassium2β-chloromethyl-2α-methylpenam-3α-carboxylate sulfone which is useful asan inhibitor of β-lactamases.

2. Description of the Prior Art

The presumed association between the resistance shown by β-lactamantibiotics to certain bacteria and the ability of those bacteria toproduce β-lactamases has led to an intensive search for β-lactamaseinhibitors. Clavulanic acid is an example of such a compound presentlyunder intensive study. Another β-lactamase inhibitor has in its acidform the structure ##STR1## and is disclosed in European PatentApplication No. 2927 published July 11, 1979.

The compound having the structure ##STR2## is disclosed in U.S. Pat.Nos. 4,036,847, 4,009,159, 3,993,646, 3,989,685 and 3,954,732.

For a corresponding sulfoxide having the 6-amino group acylated and, forexample, the structure ##STR3## see Tetrahedron Letters, 38:3303-3310(1975) and Kukolja et al., J. Org. Chem., 41, 2276-2279 (1976).

1,1-Dioxides of benzylpenicillin, phenoxymethylpenicillin and certainesters thereof have been disclosed in U.S. Pat. Nos. 3,197,466 and3,536,698, and in an article by Guddal et al., Tetrahedron Letters, No.9, 381 (1962). Harrison et al., in the Journal of the Chemical Society(London), Perkin I, 1772 (1976), have disclosed a variety of penicillin1,1-dioxides, including methyl phthalimidopenicillanate 1,1-dioxide andmethyl 6,6-dibromopenicillanate 1,1-dioxide. U.S. Pat. No. 3,544,581discloses 6-aminopenicillanic acid 1-oxide.

For a brief review of esters of penicillins, see U.S. Pat. No.3,996,236. Recent U.S. patents on esters of ampicillin and similarderivatives include U.S. Pat. Nos. 4,217,274, 4,081,546, 4,046,759,4,036,829, 3,963,704, 3,954,735, 3,951,954, 3,941,774, 3,939,180,3,931,150, 3,873,521, 3,860,579 and 3,697,507.

Recent U.S. patents on compounds (e.g. esters) or mixtures containingtwo like or unlike β-lactam derivatives include U.S. Pat. Nos.4,181,659, 3,928,595, 3,869,449 and 3,867,538.

U.S. Pat. No. 4,155,912 describes 2-penem-3-carboxylic acid compoundshaving the formula ##STR4## and esters and salts, and see also FarmdocAbstracts 82090A, 10336B and 44337B.

The compound (under the number CP-45899) having the structure ##STR5##is an irreversibly acting β-lactamase inhibitor with excellent solutionstability. It has weak antibacterial activity and potentiates the invitro and in vivo activities of ampicillin versus β-lactamase-producingstrains [A. R. English et al., Antimicrobial Agents and Chemotherapy,14, 414-419 (1978), Aswapokee et al., J. Antibiotics 31(12), 1238-1244(Dec. 1978) and Derwent's Farmdoc Abstracts 89627A and 73866B and U.S.Pat. No. 4,234,579].

Cignarella and associates [J. Org. Chem. 27, 2668 (1962)] reported thefirst deamination of 6-aminopenicillanic acid (6-APA) (1). When treatedwith sodium nitrite and hydrochloric acid, 6-APA was converted to6-chloropenicillanic acid (96), isolated as the dibenzylethylenediaminesalt. Substitution of hydrobromic acid in the reaction gave the 6-bromoderivative (97). Both deaminated products were devoid of microbiologicalactivity against M. pyogenes aureus 209P. ##STR6##

Evrard and co-workers [Nature, 201, 1124 (1964)] prepared the methylesters of (96) and (97) and showed that (97) could be hydrogenated topenicillanic acid (98). Their study emphasized the value of gaschromatography in the separation of penicillanates including methylpenicillanate (99). ##STR7##

Compound 98 above is also described in U.K. Pat. No. 1,072,108 (1967).

6β-Bromopenicillanic acid is claimed in U.S. Pat. No. 4,180,506 anddescribed as potentiating the in vitro activity of benzylpenicillin andampicillin against certain bacteria.

According to U.S. Pat. Nos. 4,203,992 and 4,203,993(2S,5R,6S)-6β-Bromo-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylicacid, S,S-dioxide, physiologically acceptable salts thereof and readilyhydrolyzable ester thereof inhibit the action of the β-lactamase enzymeRTEM.

EPO No. 13,617 states that its invention relates to a process for thepreparation of certain penicillin derivatives which are useful asβ-lactamase inhibitors. The compounds which may be prepared by theprocess of this invention have formula (1), or a pharmaceuticallyacceptable salt or ester thereof: ##STR8## wherein R representshydrogen, halogen, C₁₋₆ alkylthio, C₁₋₆ alkyl or alkyl substituted withphenyl, carboxy, C₁₋₆ -alkoxycarbonyl, hydroxy or C₁₋₆ alkylthio, and nis zero, 1 or 2. In addition certain of the compounds produced by theprocess of the invention are novel compounds. Claim 1 therein reads asfollows:

1. A compound of the formula (II): ##STR9## or a pharmaceuticallyacceptable salt or in-vivo hydrolysable ester thereof wherein X is abromine or chlorine atom, and n is zero, 1 or 2, when substantially freefrom the corresponding 6α-substituted compound.

The compound (under the number CP-45,899 and now known as sulbactam)having the structure ##STR10## is an irreversibly acting β-lactamaseinhibitor with excellent solution stability. It has weak antibacterialactivity and potentiates the in vitro and in vivo activities ofampicillin versus β-lactamase-producing strains [see Derwent's FarmdocAbstracts 89627A and 73866B].

It is disclosed by B. Baltzer et al., Mutual Pro-Drugs of β-LactamAntibiotics and β-Lactamase Inhibitors, J. Antibiotics, 33(10),1183-1192 (1980) that the principle of combining a β-lactam antibioticwith a β-lactamase inhibitor in a single molecule functioning aspro-drug for the two active components is illustrated by the linkedesters 3 and 4 in which ampicillin and mecillinam, respectively, arecombined with the β-lactamase inhibitor penicillanic acid sulfone. It isshown that in man these esters are excellently absorbed from thegastro-intestinal tract and after absorption hydrolyzed withsimultaneous liberation of the active components. As a result high bloodand tissue levels of antibiotic and β-lactamase inhibitor in a balancedratio are attained. The advantages of "mutual pro-drugs" over simplecombinations are discussed.

Esters 3 and 4 referred to therein have the structures ##STR11##

The following publications were discussed in the Baltzer et al.publication:

(1) Von Daehne, W.; E. Frederiksen, E. Gundersen, F. Lund, P. Morch, H.J. Petersen, K. Roholt, L. Tybring & W. O. Godtfredsen: Acyloxymethylesters of ampicillin. J. Med. Chem. 13: 607-612, 1970

(2) Binderup, E.; W. P. Godtfredsen & K. Roholt: Orally activecephaloglycin esters. J. Antibiotics 24: 767-773, 1971

(3) Wheeler, W. J.; W. E. Wright, V. D. Line & J. A. Frogge: Orallyactive esters of cephalosporin antibiotics. Synthesis and biologicalproperties of7-(D-2-amino-2-phenylacetamido)-3-[5-methyl-(1,3,4-thiadiazol-2-yl)thiomethyl]-3-cephem-4-carboxylicacid. J. Med. Chem. 20: 1159-1164, 1977

(4) Wheeler, W. J.; D. A. Preston, W. E. Wright, G. W. Huffman, H. E.Obsborne & D. P. Howard: Orally active esters of cephalosporinantibiotics. 3. Synthesis and biological properties ofaminoacyloxymethyl esters of7-[D-(-)-mandelamino]-3-[[(1-methyl-1Htetrazol-5-yl)thio]methyl]-3-cephem-4-carboxylicacid. J. Med. Chem. 22: 657-661, 1979

(5) Wright, W. E.; W. J. Wheeler, V. D. Line, J. A. Frogge & D. R.Finley: Orally active esters of cephalosporin antibiotics. II. Synthesisand biological properties of the acetoxymethyl ester of cefamandole. J.Antibiotics 32: 1155-1160, 1979

(6) Roholt, K.: Pharmacokinetic studies with mecillinam andpivmecillinam. J. Antimicrob. Chemother. 3 (Suppl. B): 71-81 1977

(7) Foulds, G.; W. E. Barth, J. R. Bianchine, A. R. English, D. Girard,S. L. Hayes, M. M. O'Brien & P. Somani: Pharmacokinetics of CP-45,899and pro-drug CP-47,904 in animals and humans. Current Chemother, &Infect. Disease 1980: 353, 1980

(8) Aswapokee, N. & H. C. Neu: A sulfone β-lactam compound which acts asa β-lactamase inhibitor. J. Antibiotics 31: 1238-1244, 1978

(9) Engberg-Pedersen, H: Empirical equation for pharmacokinetic analysisof drug serum levels after oral application. Antimicr. Agents & Chemoth.6: 554-562, 1974

(10) Jordan, M. C.; J. B. De Maine & W. M. M. Kirby: Clinicalpharmacology of pivampicillin as compared with ampicillin. Antimicr.Agents & Chemoth.-1970: 438-441, 1971

(11) Godtfredsen, W. O.: U.S. Pat. No. 3,869,449, 1975

(12) Christensen, B. G. & W. J. Leanza: U.S. Pat. No. 3,931,150, 1976

(13) Evrard, E.; M. Claesen & H. Vanderhaeghe: Gas chromatography ofpenicillin and penicillanic acid esters. Nature 201: 1124-1125, 1964

(14) English, A. R.; J. A. Retsema, A. E. Girard, J. E. Lynch & W. E.Barth: GP-45,899, a beta-lactamase inhibitor that extends theantibacterial spectrum of beta-lactam: Initial bacteriologicalcharacterization. Antimicro. Agents & Chemoth. 14: 414-419, 1978

It is stated in GB No. 2044255 published Oct. 15, 1980 that theinvention therein relates to hitherto unknown compounds of the generalformula 1: ##STR12## in which R₁ stands for a phenyl, 4-hydroxyphenyl,1,4-cyclohexadienyl or a 3-thienyl group; R₂ represents a primary aminoor a carboxy group; R₃ is a hydrogen atom, or a lower alkyl, aryl oraralkyl radical, and A stands for a radical of a β-lactamase inhibitorcontaining a β-lactam ring as well as a carboxy group, A being connectedvia the carboxy group.

The new compounds are useful in the treatment of bacterial infectionsand are in particular strongly active against β-lactamase producingbacteria. See also Farmdoc abstracts 60773C and 60776C.

More recent specifications or patents issued or published on or afterNovember, 1980 include the following:

U.S. Pat. No. 4,234,579 (Pfizer, Inc.--"Penicillanic Acid 1,1-Dioxidesas β-Lactamase Inhibitors");

U.S. Pat. No. 4,244,951 (Pfizer, Inc.--"Bis-Esters of Methanediol withPenicillins and Penicillanic Acid 1,1-Dioxide");

GB Pat. No. 2,045,755A (Pfizer, Inc.--"Process and Intermediates forPenicillanic Acid 1,1-Dioxide and Esters Thereof");

GB Pat. No. 2,051,046A (Leo Pharmaceutical Products Ltd.--"PenicillanicAcid Derivatives");

GB Pat. No. 059,960A (Leo Pharmaceutical Products Ltd.--"Method forProducing Penicillanic Acid Derivatives").

SUMMARY OF THE INVENTION

The present invention provides the acid having the formula ##STR13## ora pharmaceutically acceptable salt of said acid or an easily hydrolyzedester of said acid.

The pharmaceutically acceptable salts referred to above include nontoxicmetallic salts such as sodium, potassium, calcium and magnesium, theammonium salt and substituted ammonium salts, e.g. salts of suchnontoxic amines as trialkylamines (e.g. triethylamine), procaine,dibenzylamine, N-benzyl-β-phenethylamine, 1-ephenamine,N,N'-dibenzyl-ethylenediamine, dehydroabietylamine,N,N'-bis(dehydroabietyl)ethylenediamine, N-(lower)alkylpiperidine (e.g.N-ethylpiperidine) and other amines which have been used to formpharmaceutically acceptable salts of penicillins and cephalosporins. Themost preferred salts are the alkali metal salts, i.e. the sodium andpotassium salts, and the ammonium salt.

As used herein the term "physiologically hydrolyzed esters" refers tothose pharmaceutically acceptable esters known in the art to hydrolyzeto the free acid form in vivo. Examples of suitable physiologicallyhydrolyzed esters include phenacyl, acetoxymethyl, pivaloyloxymethyl,α-acetoxyethyl, α-acetoxybenzyl, α-pivaloyloxyethyl,phthalidyl(3-phthalidyl), indanyl(5-indanyl), methoxymethyl,benzoyloxymethyl, α-ethylbutyryloxymethyl, propionyloxymethyl,valeryloxymethyl, isobutyryloxymethyl,6-[(R)-2-amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carbonyloxymethyland6[(R)-2-amino-2-p-hydroxyphenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carbonyloxymethyland others for which the formulae are set forth below. The preferredesters are the acetoxymethyl, pivaloyloxymethyl, methoxymethyl,phthalidyl, 5-indanyl,6-[(R)-2-amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carbonyloxymethyland6-[(R)-2-amino-2-p-hydroxyphenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carbonyloxymethyl.

There is further provided by the present invention the process for theproduction as the desired product of the acid having the formula##STR14## or a pharmaceutically acceptable salt of said acid whichcomprises the consecutive steps of

(a) catalytically hydrogenating, as with a precious metal catalyst suchas palladium, an ester having the formula ##STR15## wherein R¹ is benzylor substituted benzyl and then (b) subjecting the hydrogenated productto oxidation to produce said desired acid or salt thereof and then, ifdesired,

(c) esterifying said acid or salt thereof to produce an easilyhydrolyzed ester of said acid.

There is further provided by the present invention the process for theproduction as the desired product of the acid having the formula##STR16## or a pharmaceutically acceptable salt of said acid whichcomprises the consecutive steps of

(a) oxidizing, as with KMnO₄, H₂ O₂ or like peroxide or peracid, anester having the formula ##STR17## to produce an ester sulfoxide havingthe formula ##STR18## and then (b) reacting said ester sulfoxide(preferably in solution in an inert organic solvent and preferably indimethylformamide) with a metal in acid, as with zinc in glacial aceticacid, at a temperature above 0° C. to produce said desired acid or saltand then, if desired,

(c) esterifying said acid or salt thereof to produce an easilyhydrolyzed ester of said acid.

A wide variety of oxidants known in the art for the oxidation ofsulfides to sulfones can be used. However, particularly convenientreagents for alkali metal permanganates, e.g. potassium permanganate,and organic peracids, e.g. 3-chloroperbenzoic acid.

Particularly useful protecting groups for R¹ are the benzyl group andsubstituted benzyl groups, especially 4-nitrobenzyl. Benzyl andsubstituted benzyl groups can be removed conveniently by catalytichydrogenation. In this case, a solution in an inert solvent of thecompound of the formula A, wherein R¹ is benzyl or substituted benzyl,is stirred or shaken under an atmosphere of hydrogen, or hydrogen mixedwith an inert diluent such as nitrogen or argon, in the presence of acatalytic amount of a hydrogenation catalyst. Convenient solvents forthis hydrogenation are lower-alkanols, such as methanol; ethers, such astetrahydrofuran and dioxan; low molecular weight esters, such as ethylacetate and butyl acetate; water; and mixtures of these solvents.However, it is usual to choose conditions under which the startingmaterial is soluble. The hydrogenation is usually carried out at atemperature in the range from about 0° to about 60° C. and at a pressurein the range from about 1 to about 100 kg./cm.². The catalysts used inthis hydrogenation reaction are the type of agents known in the art forthis kind of transformation, and typical examples are the noble metals,such as nickel, palladium, platinum and rhodium. The catalyst is usuallypresent in an amount from about 0.01 to about 2.5 weight-percent, andpreferably from about 0.1 to about 1.0 weight-percent, based on thecompound of formula A. It is often convenient to suspend the catalyst onan inert support; a particularly convenient catalyst is palladiumsuspended on an inert support such as carbon. Additionally it is usualto buffer the reaction mixture in order to operate at a pH in the rangefrom about 4 to 9, and preferably from 6 to 8. Borate and phosphatebuffers are commonly used. The reaction typically takes about one hour.

There is further provided by the present invention the esters having theformula ##STR19## wherein R is ##STR20## wherein R¹ is hydrogen orhydroxy and R² is hydrogen, hydroxy, methyl, methoxy or chloro andpreferably the ester having the formula ##STR21## and the nontoxic,pharmaceutically acceptable acid addition salts of said esters and theprocess for producing such an ester of the former type which comprisesthe treatment with acid of a solution of a compound having the formula##STR22## wherein R is ##STR23## wherein R⁴ is hydrogen or hydroxy andR⁵ is hydrogen, hydroxy, methyl, methoxy or chloro and R¹ is alkyl,aralkyl or aryl, R² is hydrogen, alkyl, aralkyl or aryl and R³ is alkyl,aralkyl, aryl, alkoxy, aralkoxy, aryloxy or ##STR24## wherein R⁴ and R⁵are each hydrogen, alkyl, aralkyl or aryl or, when taken together withthe nitrogen atom, are piperidino or morpholino with said treatment withacid preferably being carried out in an organic solvent such as acetoneor chloroform or in aqueous or partly aqueous solution and preferablybetween pH 1 and pH 5 at room temperature and the process for producingan ester of the latter type which comprises the reaction of a compoundof the formula ##STR25## wherein R has the meaning set forth above withabout an equimolar weight of the compound having the formula ##STR26##in an inert, anhydrous organic solvent, preferably dimethylformamide, ata low termperature, and preferably at about 0° C. to 10° C. untilcompletion of the reaction.

It is further preferred that, in the amino-protecting group, R¹ ismethyl, R² is hydrogen and R³ is methoxy, ethoxy or methyl; thisrequires the use of methyl acetoacetate, ethyl acetoacetate oracetylacetone.

In the removal of the α-amino-protecting group it is preferred that usebe made of a strong mineral acid such as hydrochloric acid or of formicacid.

There is further provided by the present invention as a novelintermediate an ester having the formula ##STR27## wherein R is benzylor substituted benzyl, and preferably p-nitrobenzyl, and the process forits production which comprises heating, preferably at reflux, a compoundhaving the formula ##STR28## wherein R is benzyl or substituted benzyland preferably p-nitrobenzyl, in an inert, anhydrous organic solvent,preferably dioxane, in the presence of large, and preferably equimolar,amounts of a weak tertiary amine, preferably quinoline, and an acidchloride, preferably benzoyl chloride, until the reaction issubstantially complete.

There is provided by the present invention in addition as a novelintermediate an ester having the formula ##STR29## wherein R is benzylor substituted benzyl, and preferably p-nitrobenzyl, and the process forits production which comprises oxidizing a solution in an inert solvent,preferably methylene chloride, of a compound having the formula##STR30## wherein R is benzyl or substituted benzyl, and preferablyp-nitrobenzyl, at about room temperature by the use of a peracid,preferably m-chloroperoxybenzoic acid.

There is further provided by the present invention as a novelintermediate the ester having the formula ##STR31## the process for itsproduction which comprises oxidizing a solution in an inert solvent,preferably methylene chloride, of a compound having the formula##STR32## at about room temperature by the use of an oxidizing agentsuch as KMnO₄, H₂ O₂ or like peroxide or, preferably a peracid,preferably m-chloroperoxybenzoic acid.

"Skellysolve B" is a petroleum ether fraction of b.p. 60°-68° C.consisting essentially of n-hexane ("Skellysolve" is a trade name of theSkelly Oil Co.).

The following examples are provided solely for the purpose ofillustrating the preparation of representative compounds of the presentinvention and are not to be construed as limiting the invention.

EXAMPLE 1 Preparation of Potassium2β-Chloromethyl-2α-methylpenam-3α-carboxylate Sulfone (BL-P2013)##STR33## 6α-Bromopenicillanic Acid S-Sulfoxide (1)

1. Dissolve 30 g (37.5 mmole) 6α-bromopenicillanic acidN,N'-dibenzylethylenediamine salt [G. Cignarella et al., J. Org. Chem.27, 2668 (1962) and E. Evrard, Nature 201, 1124 (1964)] in 330 ml ofmethylene chloride. Agitate and cool to 0° C.

2. Slowly add 13 ml (156 mmole) concentrated hydrochloric acid into themethylene chloride solution. The precipitation ofdibenzylethylenediamine HCl salt (DBED.HCl) takes place within a minute.Stir the slurry at 0°-5° C. for 10 minutes.

3. Filter to remove the DBED.HCl precipitate through a precoateddiatomaceous earth ("Dicalite") filter. Wash the cake with 150 ml ofmethylene chloride. The filtration should be completed as quickly aspossible. Avoid holding the acidic methylene chloride solution for aprolonged period. There may be some filtration problems because of thefine nature of the precipitate. Addition of filter aid to the slurry maybe helpful.

4. Wash the combined methylene chloride filtrates with 60 ml of coldwater. Agitate 5 minutes and discard the aqueous phase. The pH of thewash is 2.0-2.3.

5. The methylene chloride solution containing 6α-bromopenicillanic acidis concentrated under reduced pressure to a volume of 65-80 ml. Cool andstir the solution to 5° C.

6. With vigorous agitation cautiously add 13 ml (86.9 mmoles) of 40%peracetic acid over a period of 30 minutes. The reaction is exothermic.Maintain temperature between 15°-18° C. with ice bath cooling. Thesulfoxide begins to crystallize after 10 ml peracetic acid is added.Cool and stir the slurry at 0°-5° C. for two hours.

7. Filter and wash the snow white cake with the following sequence: 10ml 5° C. water, then 10 ml 0°-5° C. methylene chloride, and finally washwith 15 ml of heptane.

8. Dry the cake in 45° C. air oven to constant weight, about 6-10 hoursshould be sufficient. Extended heating may generate a trace of pinkishcolor. The weight of 1 is about 16.26 gm, 73.24% yield.

9. The reaction mix and final product may be monitored by TLC using 15toluene/4 acetone/1 acetic acid (HOAC) or 8 acetone/8 methanol/3toluenel/HOAC solvent systems. The final product should be analyzed byNMR and IR.

p-Nitrobenzyl 6α-Bromopenicillanate S-Sulfoxide (2)

To a solution of 12 g (0.04 mole) of 6α-bromopenicillanic acidS-sulfoxide in 100 ml of acetone was added 7.5 g (0.041 mole) ofpotassium 2-ethylhexanoate. The salt was collected by filtration, washedwith cold acetone and air dried to yield a total of 10 grams. Thecrystalline potassium salt was dissolved in 75 ml of dimethylacetamideand 7.8 g (0.04 mole) of p-nitrobenzyl bromide was added. The solutionwas stirred at 23° C. for 24 hours. The mixture was diluted with 500 mlof water and extracted with ethyl acetate. The ethyl acetate layer waswashed four times with water and dried over anhydrous magnesium sulfate.The solvent was evaporated at 35° C. (15 mm) to an oil whichcrystallized. The light tan crystals of 2 were slurried with ether andcollected by filtration to yield 9 g (70%) mp 124°-125° C. dec.

Anal. Calc'd for C₁₅ H₁₅ BrN₂ O₆ S: C, 41.98; H, 3.05; N, 6.52; Found:C, 42.00; H, 3.48; N, 6.98.

IR(KBr): 1800(s), 1740(s), 1610(w), 1520(s), 1450(m), 1350(s), 1060(m),740(m) cm⁻¹. H-NMR (60 mHz, DMSO): δ1.22 (s,3H), 1.6 (s,3H), 4.67(s,1H), 5.2 (d,J˜1-5 Hz,1H), 5.45 (s,2H), 5.68 (d,J˜1-5 Hz,1H), 7.5-8.5(m,4H).

p-Nitrobenzyl 2β-Chloromethyl-2α-methyl-6-bromopenam-3α-carboxylate (3)

A solution of 5 g (0.012 mole) of p-nitrobenzyl 6α-bromopenicillanateS-sulfoxide (2) in 120 ml anhydrous dioxane was heated at reflux undernitrogen for 4 hours with 1.5 g (0.012 mole) of quinoline and 1.6 g(0.012 mole) of benzoyl chloride. The solution was diluted with 600 mlof water and extracted with ethyl acetate. The ethyl acetate extract waswashed with 5% sodium bicarbonate solution, 5% phosphoric acid solutionand finally with water. The organic layer was dried over anhydrousmagnesium sulfate and evaporated to an oil at 35° C. (15 mm). The oilcrystallized and was collected and washed with ether and then with coldtoluene to yield 3, 3.5 g (65%) mp 130°-135° C. dec.

Anal. Calc'd for C₁₅ H₁₅ ClBrN₂ O₅ S: C, 40.06; H, 3.14; N, 6.23. Found:C, 40.19; H, 3.12; N, 6.75.

IR(KBr): 1792(s), 1740(s), 1610(w), 1520(s), 1353(s), 1280(m), 1025(w),990(w), 750(w) cm⁻¹. NMR (60 mHz, DMSO): δ1.45 (s,3H), 3.5-4.3 (m,2H),5.05 (s,1H), 5.42 (s,2H), 5.5 (d,J˜1.5 Hz,1H), 5.62 (d,J˜1.5 Hz,1H),7.5-8.5 (m,4H).

p-Nitrobenzyl 2β-Chloromethyl-2α-methylpenam-6α-carboxylate Sulfoxide(4)

A solution of 1 g (0.0022 mole) of p-nitrobenzyl3β-chloromethyl-2α-methyl-6α-bromopenam-3α-carboxylate (3) dissolved in50 ml of methylene chloride was stirred with 473 mg of (0.0022 mole) ofm-chloroperoxybenzoic acid. The solution was stirred at 23° C. for 3hours. The methylene chloride was evaporated to 20 ml at 15 mm and 33°C. and the concentrated solution was diluted with 50 ml of heptane("Skellysolve B"). The solvent was decanted and the residue was slurriedwith ether and (4) soon crystallized; yield 250 mg, 24% mp 136°-137° C.dec.

Anal. Calc'd for C₁₅ H₁₄ BrClN₂ O₆ S: C, 38.68; H, 3.02; N, 6.02. Found:C, 39.14; H, 3.13; N, 5.96.

IR(KBr): 1800(s), 1760(s), 1520(s), 1350(s), 1200(s), 1050(m), 830(w),740(w) cm⁻¹. H-NMR (60 mHz, DMSO): δ1.32 (s,3H), 3.8-4.5 (m,2H), 4.97(s,1H), 5.25 (d,J˜1.5 Hz,1H), 5.45 (s,2H), 5.6 (d,J˜1.5 Hz,1H), 7.8-8.5(m,4H).

Potassium 2β-Chloromethyl-2α-methylpenam-3α-carboxylate Sulfone (5)(BL-P2013)

To a solution of 7 g (0.015 mole) of p-nitrobenzyl2β-chloromethyl-2α-methyl-6α-bromopenam-3α-carboxyate sulfoxide (4) in150 ml of ethyl acetate was added a suspension of 4 g of 30% palladiumon diatomaceous earth ("Celite") and 2.8 g of sodium bicarbonate in 150ml of water. The mixture was hydrogenated for 3 hours at 50 psi. Thecatalyst was separated by filtration and the aqueous layer was separatedand treated with 1.5 g of potassium permanganate in 50 ml of water. Themixture was stirred for 1 hour and 250 mg of sodium bisulfite was added.The mixture was filtered and the filtrate was adjusted to pH 2 withconcentrated hydrochloric acid. The solution was lyophilized to give awhite amorphous powder. The solid was extracted into ethyl acetate,evaporated to a volume of 20 ml and diluted with 100 ml of heptanes("Skellysolve B"). White, hygroscopic, solid2β-chloromethyl-2α-methylpenam-3α-carboxylic acid sulfone was collected.The acid was dissolved in acetone and treated with solid potassium2-ethylhexanoate. A crystalline white salt precipitated to give, aftercollection by filtration, 170 mg of 5, mp >140° C. dec.

Anal. Calc'd for C₈ H₇ ClKNO₅ S.2H₂ O: C, 28.27; H, 3.24; N, 4.12.Found: C, 28.27; H, 3.69; N, 3.84.

IR(KBr): 1790(s), 1770(m), 1620(s), 1460(m), 1370(s), 1310(s), 1200(s),1140(s), 955(m), 740(m) cm⁻¹. H--NMR (100 mHz, D₂ O): δ1.68(s,3H),3.2-3.9 (m,J˜2 Hz, J˜4 Hz, J˜6 Hz,2H), 4.0-4.4 (m,2H), 4.3 (s,1H), 5.02(d d, J˜4 Hz), J˜2 Hz,1H).

EXAMPLE 2 Pivaloyloxymethyl2β-Chloromethyl-2α-methylpenam-3α-carboxylate Sulfone

2β-Chloromethyl-2α-methylpenam-3α-carboxylic acid sulfone indimethylformamide is treated with one equivalent of triethylamine andstirred to effect solution. Bromomethyl pivalate (1 equivalent) indimethylformamide is then added. The resulting mixture is stirred atroom temperature. The mixture is then clarified by filtration and thefiltrate poured into ice water. The separated solid is collected byfiltraton, washed with water and dried to give the title ester.

The respective acetoxymethyl, methoxymethyl, acetonyl and phenacylesters of the same acid are prepared by substituting in the method abovefor the bromomethyl pivalate used therein an equimolar weight ofchloromethyl acetate, chloromethyl methyl ether, chloroacetone andphenacyl bromide, respectively.

EXAMPLE 3 Pivaloyloxymethyl2β-Chloromethyl-2α-methylpenam-3α-carboxylate Sulfone BL-P2024 ##STR34##

To a stirred suspension of 14.6 l g (0.0487 mol) of BL-P2013 (5) in 200ml of acetone was added 4 ml of a 10% aqueous solution of sodium iodideand the mixture was brought to reflux on the steam bath. To thisrefluxing suspension was added 14.8 ml (0.1 mol) or redistilledchloromethyl pivalate (B.P. 34° C. at 7 mm Hg) all at once. The mixturewas stirred at reflux for three hours and then cooled to roomtemperature (22° C.). The crystalline solids were collected byfiltration, washed with 3×30 ml of acetone and the combined filtrateswere evaporated in an oil in vacuo at <22° C. The oil was then taken upin 500 ml of ethyl acetate and washed once with water (200 ml) and oncewith saturated Na₂ SO₄ while being stirred with 2 g. of decolorizingcarbon with cooling (ice bath). After 20 minutes the mixture wasfiltered through a diatomaceous earth (Dicalite) pad with suction andthe pad was washed with 4×100 ml of ethyl acetate. The combinedfiltrates were concentrated in vacuo at 22° C. to an oil. The oil wasthen concentrated further at about 22° C. and <1 mm Hg to remove most ofthe residual chloromethyl pivalate. The remaining oil was thentriturated twice with 50 ml portions of n-pentane and then left over theweekend in the cold room (about 10° C.) under n-pentane. The solidcrystalline mass was then broken up to a solid powder under 40 ml of 4:1mixture of ether-n-pentane. The product was then collected byfiltration, washed with ether-pentane (1:1) then pentane and air dried.After drying in vacuo for four hours over P₂ O₅ there was obtained 13.37g. of pivaloyloxymethyl 2β-chloromethyl-2α-methylpenam-3α-carboxylatesulfone (about 75% yield) M.P. 93°-95° C.

Anal. Calcd. for C₁₄ H₂₀ ClNO₇ S: C, 44.03; H, 5.27; N, 3.67. Found: C,44.11; H, 5.08; N, 3.85.

EXAMPLE 4 Recrystallization of Potassium2β-Chloromethyl-2α-methylpenam-3α-carboxylate Sulfone (BL-P2013)

To a mixture of 20 ml of n-butanol and one gram of BL-P2013 (5) wasadded water, one ml at a time, with shaking in a separatory funnel untila pale yellow solution was obtained. The clear solution was filteredthrough a fluted filter paper and the flask and filter paper washed withabout 10 ml of 9:1 n-butanol-H₂ O and the combined filtrates werediluted with a further 20 ml of n-butanol. The resulting solution wasplaced in a round-bottomed flask on the roto-vap and evaporated underreduced pressure to approximately one half the original volume. The snowwhite crystalline product was collected by filtration, washed with 6×10ml of acetone and air dried. Yield 810 mg. After vacuum drying 6 hoursover P₂ O₅ at <1 mm Hg. there was obtained 800 mg M.P. 215° C. (dec.)(80% yield)

Anal. Calcd. for C₈ H₉ ClNO₅ SK.1H₂ O: C, 29.67; H, 3.39; N, 4.63; Cl,10.94; K.F.H₂ O, 5.56. Found: C, 29.23; H, 3.38; N, 4.49; Cl, 10.74;K.F.H₂ O, 5.74.

This recrystallization procedure produces a crystalline monohydratediffering from the starting material which is essentially anhydrous.

EXAMPLE 5 ##STR35## See Chemical Abstracts 27, 2427¹ and 22, 382⁸ ; andGB 299064. ##STR36##

(a) A solution of chloromethyl chlorosulfate (0.115 mol) in 40 ml.dichloromethane is added dropwise, keeping the reaction temperaturebelow 30° C., to a solution of compound 5 (0.1 mol) and potassiumbicarbonate (0.3 mol) and tetrabutylammonium hydrogen sulfate (0.01 mol)in 200 ml. dichloromethane-water (1:1). At the end of the addition themixture is stirred at room temperature for 30 minutes, the organic phaseis separated and the aqueous phase is extracted with dichloromethane (50ml.). The combined organic phases are dried (Na₂ SO₄) and evaporated invacuo to give a residue which is dissolved in ether (150 ml.). Insolublematerial is filtered off after adding diatomaceous earth and thefiltrate is evaporated in vacuo to provide compound 7.

(b) To a suspension of compound 5 (1.5 g.) in dimethylformamide (12 ml.)there is added 1.6 g. bis-chloromethyl sulfate and the mixture isstirred at room temperature for 45 minutes. After dilution with ethylacetate (50 ml.) the mixture is washed with water and then aqueoussodium bicarbonate, dried and evaporated in vacuo to leave compound 7 asan oil.

(c) To a solution of compound 5 (0.005 mol) in dimethylformamide (7.5ml.) there is added triethylamine (0.007 mol) and chloroiodomethane(0.030 mol) and the mixture is stirred at room temperature for fourhours. After dilution with ethyl acetate (30 ml.) the mixture is washedwith water (3×10 ml.) followed by saturated aqueous sodium chloride (5ml.), dried and evaporated in vacuo to leave compound 7 as an oil.

(d) To a mixture of compound 5 (0.15 mol) silver nitrate (0.15 mol) andsilver oxide (7.5 g.) in acetonitrate (750 ml.) there is addedchloroiodomethane (1.5 mol). After stirring for 48 hours at roomtemperature, the silver salts are filtered off and the filtrate isevaporated to dryness in vacuo. The residue is dissolved in ethylacetate (200 ml.) and the solution is washed with saturated aqueoussodium chloride, filtered, dried and evaporated in vacuo to givecompound 7.

Compound 7 and other intermediates and final products of the presentinvention are purified, if desired, by column chromatography, as on"Sephadex" LH20 using chloroform-hexane, 65:35 as the eluent for exampleor by silica gel chromatography, e.g. using Mallinckrodt CC-7 andhexane-ethyl acetate, 3:2 or ethyl acetate-petroleum ether, 8:2 or 7:3or 1:9 or 15:85 or ethyl acetate-n-hexane, 4:6 or 3:1 hexane-ethylacetate, 3:1 or 1:1 or 1:4 or cyclohexane-ethyl acetate, 1:1.

Thin layer chromatography is also useful. "Sephadex" is cross-linkeddextran 2-(diethylamino) ethyl2-[[2-(diethylamino)ethyl]diethylammonio]ethyl ether chloridehydrochloride epichlorhydrin (See Merck Index, Ninth Edition, itemnumber 7337).

A solution of compound 7 (0.2 mol) and sodium iodide (0.3 mol) inacetone (150 ml.) is stirred at room temperature for 18 hours. Theresulting suspension is cooled to about 0° C. and adjusted to about pH7.2 by the addition of saturated aqueous sodium bicarbonate withstirring. After decolorizing by titration with 0.5 M aqueous sodiumthiosulfate, water (150 ml.) is added dropwise to the stirred mixture toprecipitate solid compound 8 which is collected by filtration, washedwith acetone-water 1:1 (2×20 ml.), isopropanol (2×20 ml.) and ether(2×20 ml.) and dried.

Ampicillin is converted to compound 9 by the use of methyl acetoacetatein the procedures of U.S. Pat. No. 3,316,247. Then to a stirred solutionof compound 9 (0.57 mol) in dimethylformamide (1 liter) there is addedat 5° C. 0.5 mol of compound 8. After stirring for 15 minutes at 5° C.the reaction mixture is poured into an ice-cold mixture of ethyl acetate(4 liters) and saturated aqueous calcium chloride (2 liters) withstirring. The organic layer is separated, washed with saturated aqueouscalcium chloride (2×500 ml.), filtered and evaporated to about one literin vacuo to provide a concentrated solution of compound 10. To thisconcentrate there is added water (500 ml.) and n-butanol (500 ml.) andthen, dropwise, 4 N hydrochloric acid with stirring until theamino-protecting group is removed to provide a solution of compound 11.After the addition of the acid is finished ether (1 liter) and water(500 ml.) are added to the stirred mixture, the aqueous phase isseparated and the organic phase is extracted with water (800 ml.). Thecombined aqueous extracts are washed with ether (1 liter) and thensodium chloride (640 g.) and dichloromethane (2 liters) are added andthe mixture is stirred for 15 minutes. The organic phase is separatedand the aqueous phase is extracted with dichloromethane (1 liter) andthe combined organic extracts are dried (MgSO₄) and evaporated to about600 ml. under reduced pressure to give a concentrated solution ofcompound 11. Addition to the concentrate of 200 ml. 2-butanone followedby cooling precipitates solid6-[(R)-2-Amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carbonyloxymethyl2β-chloromethyl-2-α-methylpenam-3α-carboxylate sulfone (11) which iscollected by filtration.

EXAMPLE 66-[(R)-2-Amino-2-p-hydroxyphenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carbonyloxymethyl2β-chloromethyl-2-α-methylpenam-3α-carboxylate sulfone having theformula ##STR37## is produced by substituting amoxicillin for theampicillin used in the procedure of Example 5. EXAMPLE 7 ##STR38##

As taught by U.S. Pat. No. 3,860,579, recrystallized phthalide (50 g.,0.375 mol) and recrystallized N-bromosuccinimide (0.375 mol) wererefluxed 4.5 hours in the presence of about 100 mgm α-azobutyronitrilein one liter CCl₄. The mixture was cooled to about 15° C. and filteredto remove succinimide which was itself washed with about 100 ml. CCl₄and filtered. The combined CCl₄ phases were concentrated in vacuo toabout 150 ml. giving solid 3-bromophthalide which was collected byfiltration, washed with about 50 ml. CCl₄ and air-dried to yield 54 g.which weighed 50 g. after recrystallization from boiling cyclohexane,m.p. 84°-86° C. ##STR39##

To a stirred partial solution and partial suspension of compound 5(BL-P2013; 2.3 g., 0.0075 mol) in 20 ml. dimethylformamide (DMF; driedat least 3 weeks over 3A° molecular sieves was added 1.7 g. (0.008 mol)of 3-bromophthalide (12) and the mixture was stirred 4 hours at 22° C.The resulting mixture was poured into a mixture of 200 ml. ice-coldwater and 200 ml. ice-cold ethyl acetate (rinsing the flask with alittle ethyl acetate) and the mixture was shaken. Then the organicsolvent phase was separated and washed with seven portions of ice-coldwater (100 ml.). The ethyl acetate phase was washed once with saturatedaqueous Na₂ SO₄, dried in the cold over Na₂ SO₄, filtered and evaporatedto dryness in vacuo to leave as the residue an oil which was trituratedtwice with methylcyclohexane (25 ml.), twice with "Skellysolve B" (b.p.60°-68° C., essentially n-hexane) (25 ml.) and four times with 25 ml.n-hexane to 2.5 g. compound 13 as a nearly white solid after drying inair. This product was then dried over P₂ O₅ at less than 1 mm Hg to give2.5 g. compound 13, m.p. 104° C. (dec.). Its estimated purity was85-95%.

Anal. Calcd. for C₁₆ H₁₄ ClNO₇ S: C, 51.61; H, 3.79; N, 3.77; Cl, 9.53.Found: C, 52.59; H, 4.67; N, 3.21; Cl, 7.73; K.F.H₂ O, 0.27.

EXAMPLE 8 Pivaloyloxymethyl2β-Chloromethyl-2α-methylpenam-3α-carboxylate Sulfone

A mixture of 1 g. (0.0031 mole) of potassium2β-chloromethyl-2α-methylpenam-3α-carboxylate sulfone hydrate and 1 g.of 3A molecular sieves was stirred in 15 ml. of dimethylacetamide for 2hours at 23°. To this mixture was added 470 mg. (0.0031 mole) ofpivaloyloxymethyl chloride and the stirring was continued for 18 hours.The molecular sieves were collected and the filtrate was diluted with100 ml. of water and extracted with ethyl acetate. The ethyl acetate waswashed nine times with water and dried over anhydrous magnesium sulfate.The solvent was removed at 30° (15 mm) to leave an oil which waschromatographed on silica using silicar CC-7 (methylene chloride 8,ethyl acetate 2) showing 1 spot Rf. 0.5. The residue obtainedcrystallized from heptane ("Skellysolve B") to yield 100 mg. (M.P.94°-95°) of pivaloyloxymethyl2β-chloromethyl-2α-methylpenam-3α-carboxylate sulfone.

Anal. Calcd.: C, 44.03; H, 5.27; N, 3.67. Found: C, 44.20; H, 5.24; N,3.63.

The NMR and IR spectra were consistent for structure.

EXAMPLE 9 Sodium 2β-Chloromethyl-2α-methylpenam-3α-carboxylate Sulfone##STR40##

To a stirred solution of 500 mg. of BL-P2013 (potassium salt) in 5 ml.of H₂ O and 10 ml. of ethyl acetate was added 2 N HCl until pH 1 wasobtained (done in an ice-bath with vigorous stirring). The mixture wasthen saturated with Na₂ SO₄, the aqueous layer separated and the organicphase dried briefly in ice over Na₂ SO₄, filtered and treated dropwisewith 50% NaEH (sodium 2-ethylhexanoate) in anhydrous n-butanol untilneutral to moist pH paper. Product did not crystallize upon scratchingand was then concentrated in vacuo to an oil which was dissolved inacetone (5 ml.), scratched--no crystals, ether added to cloud point--nocrystals. Concentrated in vacuo on rotovap. to an oil which wasdissolved in ethyl acetate--added one drop H₂ O--scratched--no crystals.Concentrated in vacuo and then residue was triturated with 5 ml. ofn-butanol--200 mg. of amorphous white powder obtained, ether washed--airdried--vacuum dried over P₂ O₅ for 24 hours. 180 mg. final yield ofsodium 2β-chloromethyl-2α-methylpenam-3α-carboxylate sulfone; dec.pt. >100° indef.

Anal. Calcd. for C₈ H₉ ClNO₅ SNa: C, 33.10; H, 3.13; N, 4.89. Found: C,33.20; H, 3.69; N, 4.44 K.F.H₂ O, 4.04.

EXAMPLE 10 Potassium 2β-chloromethyl-2-methylpenam-3-carboxylate Sulfone(BL-P2013)

To 10 L of water, 130 g. (1.25 mole) of sodium hydrogen carbonate and200 g. of 10% Pd on BaSO₄ were added 272 g. (0.565 mole) ofp-nitrobenzyl 6α-bromo-2β-chloromethyl-2-methylpenam-3-carboxylatesulfone dissolved in 5 L of ethyl acetate. The mixture was hydrogenatedat 40° C. and 1 Kg of pressure. After 5 hours, the hydrogen uptakebecame very slow and 200 g of 10% Pd on BaSO₄ were added and the mixturehydrogenated until no further significant hydrogen absorption wasperceptible.

The slurry was filtered through a diatomaceous earth ("Celite") pad, thepad was washed with water and the aqueous phase washed with 3 L of ethylacetate. To the aqueous solution, 3 L of ethyl acetate were added andthe pH of the mixture adjusted to 1.5 with 150 ml of 12 N HCl at 10° C.The organic phase was separated and the aqueous solution saturated withNa₂ SO₄.10 H₂ O and extracted with 2×1 L of ethyl acetate. The combinedextracts were dried with magnesium sulfate. The drying agent was removedand 260 ml of 2 N potassium 2-ethylhexanoate in butanol were added at 0°C.

After stirring 2 hours at 0° C., the potassium2β-chloromethyl-2-methylpenam-3-carboxylate sulfone (BL-P2013) wascollected and dried in a vacuum at room temperature.

Yield: 134.8 g (about 70%).

EXAMPLE 11 p-Nitrobenzyl 6α-Bromopenicillinate Sulfoxide ##STR41##Procedure

To 200 ml. of N,N-dimethylacetamide was added 44 g. (0.148 mole) of6α-bromopenicillanic acid sulfoxide followed by 20.5 ml. (0.148 mole) oftriethylamine and 38.2 g. (0.177 mole) of p-nitrobenzyl bromide. It wasstirred at 22° for 20 hours.

The reaction mixture was poured into 1 liter H₂ O and extracted into3×300 ml. of methylene chloride. The combined methylene chlorideextracts were washed with 200 ml. of 5% aqueous sodium bicarbonatesolution and dried over sodium sulfate at 5° for half an hour. Thesolution was filtered and evaporated under vacuum to a residue. Theresidue was diluted with ether and the solid collected by filtration toyield 54 g. p-nitrobenzyl 6α-bromopenicillinate sulfoxide after drying.

85% yield.

nmr consistent for structure.

The yield for this step was the same as for the K-salt esterification.The advantage is there was no need to make the K-salt. (A step whichgoes in 85% to 90% yield).

EXAMPLE 12 Preparation of p-Nitrobenzyl 6α-Bromopenicillanate Sulfoxide

To 4.375 L of N,N-dimethylacetamide was added 873.0 g (2.95 moles) of6α-bromopenicillanic acid (S) sulfoxide and then with stirring and whilekeeping the internal temperature below 35° C., 293 g (2.95 moles) oftriethylamine followed by 764 g (3.54 moles) of p-nitrobenzyl bromide.The mixture was stirred then at room temperature for 5 hours and letstand overnight.

The reaction mixture was poured into 20 L of water and extracted with3×7 L of methylene chloride. The combined organic extracts were washed5×7 L of water and then with 7 L of 5% aqueous sodium bicarbonatesolution and dried over anhydrous magnesium sulfate.

The magnesium sulfate was filtered off and the solution evaporated to acrystalline residue; 4 L of diethyl ether were added and the crystalscollected to yield after drying at room temperature 1171 g (92%) ofp-nitrobenzyl 6α-bromopenicillanate sulfoxide.

Br 18.48% (calculated 18.53%), α_(D) (0.25% MeOH) +162°.

EXAMPLE 13 Preparation of p-Nitrobenzyl6α-Bromo-2β-chloromethyl-2-methylpenam-3-carboxylate Sulfone

To 16 L of acetic acid was added 364.6 g (0.812 mole) of p-nitrobenzyl6α-bromo-2β-chloromethyl-2-methylpenam-3-carboxylate. To the solution soobtained and stirred at room temperature, a solution of 282 g (1.78mole) of KMnO₄ in 26 L of water was added dropwise over 3 hours. Themixture was then stirred at room temperature for 1 hour and H₂ O₂ (37%)was added dropwise until a colorless solution was obtained. 30 L ofwater were then added, the mixture stirred for 1 hour at roomtemperature and the crystalline precipitate was collected, washed with3×5 L of water and with 2×2 L of ethanol and dried over vacuum at roomtemperature.

Yield: 297 g (76%).

α_(D) (0.5% CH₂ Cl₂) +75.9°.

EXAMPLE 14 Preparation of BL-P2013 Free Acid ##STR42##

To a mixture of 25 ml of ethyl acetate and 10 ml of water was added 800mg (0.00261 mole) of BL-P2013 potassium salt. After all of the solid haddissolved, the mixture was treated dropwise with 50% aqueous phosphoricacid with vigorous shaking until no more material precipitated from theaqueous layer. The ethyl acetate layer was separated, then washed withsaturated sodium chloride solution and dried over anhydrous magnesiumsulfate. The drying agent was removed by filtration and washed with 10ml of ethyl acetate. (The wash solvent was combined with the originalfiltrate). "Skellysolve B" was then added to the ethyl acetate to thecloud point (approx. 10 ml). The mixture was treated with 500 mg ofactivated carbon ("Darko KB") and filtered. The filtrate was dilutedwith 15 ml of "Skellysolve B", then seeded with crystals of BL-P2013free acid. After approx. 3 hours at room temperature, the crystallineprecipitate of free acid was collected and dried in vacuo (15 min) overP.sub. 2 O₅ to obtain 323 mg (46%) m.p. slow decomp. over 100°.

Anal. Calcd. for C₈ H₁₀ ClNO₅ S: C, 35.89; H, 3.77; N, 5.23, Cl, 13.25.Found: C, 35.88, H, 3.91; N, 5.41; Cl, 13.52.

This product was found to be unstable when stored at 23° C. for sevendays.

EXAMPLE 15 6α-Bromopenicillanic Acid Sulfoxide ##STR43##

To 3 l of methylene chloride was added 300 g (0.75 mole) of6α-bromopenicillanic acid N,N'-dibenzylethylenediamine salt and thissuspension was cooled to 5°. Then over a 15 min. period, with goodstirring, 130 ml of conc. HCl was added dropwise. The slurry was stirredat 5° for 2 hours. It was then filtered through a ("Celite") pad ofdiatomaceous earth and the cake was washed with 3×250 ml of methylenechloride.

The combined methylene chloride solutions were washed with 2×500 ml H₂ Oand dried over sodium sulfate for 15 min. The sodium sulfate was removedby filtration and the filtrate evaporated under reduced pressure toapprox. 750 ml.

This solution was cooled to 5° and, with vigorous stirring, 130 ml of40% peracetic acid was added dropwise such that the temperature wasmaintained at 5° to 12°. The addition was quite exothermic. At the endof the addition, the slurry was stirred at 5° for 2 hours and theproduct collected by filtration and washed with 100 ml of cold H₂ O (5°)and 100 ml of cold methylene chloride (5°). There was obtained 126 g(57%) of 6α-bromopenicillanic acid sulfoxide, m.p. 129°. The ir and nmrspectra were consistent for the desired product.

Anal. Calcd. for C₈ H₁₀ BrNO₄ S: C, 32.44, H, 3.40; N, 4.73. Found: C,32.30; H, 3.35; N, 4.71; H₂ O, 2.18.

Potassium 6α-Bromopenicillanate Sulfoxide ##STR44##

To 3 l of acetone was added 126 g (0.43 mole) of 6α-bromopenicillanicacid sulfoxide and 162 ml of 50% by weight potassium 2-ethylhexanoate inn-butanol. After stirring 1 hour at 22°, the product was collected byfiltration, washed with 2×250 ml of acetone and dried. There wasobtained 127 g (90%) of potassium 6α-bromopenicillanate sulfoxide, m.p.185°. The ir and nmr spectra were consistent for the desired structure.

Anal. Calcd. for C₈ H₉ BrKNO₄ S: C, 28.75; H, 2.71; N, 4.19. Found: C,29.03; H, 2.78; N, 4.04.

p-Nitrobenzyl 6α-Bromopenicillanate Sulfoxide ##STR45##

To 1 l of N,N-dimethylacetamide was added 145 g (0.43 mole) potassium6α-bromopenicillanate sulfoxide, and, with stirring, there was thenadded 115 g (0.53 mole) of p-nitrobenzyl bromide at 22°. The mixture wasstirred at 22° for 20 hours.

The reaction mixture was poured into 3 l of H₂ O and extracted with3×1500 ml of ethyl acetate. The combined ethyl acetate extracts werewashed with 2×500 ml of 5% aqueous sodium bicarbonate solution and driedover sodium sulfate for 1/2 hour. The sodium sulfate was filtered offand the filtrate evaporated under reduced pressure to a residue to which1 l of diethyl ether was added causing the product to crystallize. Thecrystals were collected by filtration, washed with 2×100 ml of diethylether and dried to yield 162 g (87%) of p-nitrobenzyl6α-bromopenicillanate sulfoxide, m.p. 111°. The ir and nmr spectra wereconsistent for the desired structure.

Anal. Calcd. for C₁₅ H₁₆ BrN₂ O₆ S: C, 41.78; H, 3.51; N, 6.50. Found:C, 41.66; H, 3.45; N, 6.85; H₂ O, 0.69.

p-Nitrobenzyl 6α-Bromo-2β-chloromethyl-2-methylpenam-3-carboxylate##STR46##

To 1 l of p-dioxane was added to 70 g (0.16 mole) of p-nitrobenzyl6α-bromopenicillanate sulfoxide followed by 21.2 ml (0.10 mole) ofbenzoyl chloride and 21.8 ml (0.19 mole) of quinoline. The reactionmixture was refluxed for 4 hours and then cooled to 22°, poured into2500 ml of H₂ O and extracted into 3×800 ml of ethyl acetate. Thecombined ethyl acetate extracts were washed with 300 ml of 5% aqueoussodium bicarbonate solution, 300 ml of 5% aqueous phosphoric acid and300 ml of H₂ O. The ethyl acetate solution was dried over sodium sulfatefor 1/2 hour and the sodium sulfate was removed by filtration. Thefiltrate was evaporated under reduced pressure to a residue which wasredissolved in 1 l. of ethyl acetate and again evaporated under reducedpressure to a residue. Then 1 l. of diethyl ether was added and theproduct collected by filtration to yield 41 g (57%) of p-nitrobenzyl6α-bromo-2β-chloromethyl-2-methylpenam-3-carboxylate, m.p. 132°. The irand nmr spectra were consistent for the desired structure.

Anal. Calcd. for C₁₅ H₁₄ BrClN₂ O₅ S: C, 40.06; H, 3.14; N, 6.23. Found:C, 40.62; H, 3.11; N, 6.13.

p-Nitrobenzyl 6α-Bromo-2β-chloromethyl-2-methylpenam-3-carboxylateSulfoxide ##STR47##

To 1200 ml of methylene chloride was added 51 g (0.11 mole) ofp-nitrobenzyl 6α-bromo-2β-chloromethyl-2-methlypenam-3-carboxylatefollowed by 23 g (0.12 mole) of m-chloroperoxybenzoic acid. The solutionwas stirred at 22° for 2 hours and evaporated under reduced pressure toa wet residue. The residue was stirred with 4 l of diethyl ether for 1hour and allowed to stand at 10° for 20 hours. The product crystallizedout and was collected by filtration, washed with 2×200 ml of diethylether and dried, yielding 39 g p-nitrobenzyl6α-bromo-2β-chloromethyl-2-methylpenam-3-carboxylate sulfoxide (75%),m.p. 132°. The ir and nmr spectra were consistent for the desiredstructure.

Anal. Calcd. for C₁₅ H₁₄ BrClN₂ O₆ S: C, 38.69; H, 3.03; N, 6.07. Found:C, 38.98; H, 3.04; N, 5.84; H₂ O, 0.35.

Potassium 2β-Chloromethyl-2-methylpenam-3-carboxylate Sulfone (BL-P2013)##STR48##

To 600 ml of H₂ O was added 8 g of 30% Pd on "Celite" and 16 g (0.19mole) of sodium bicarbonate. Then 32 g (0.69 mole) of p-nitrobenzyl6α-bromo-2β-chloromethyl-2-methylpenam-3-carboxylate sulfoxide wasdissolved in 400 ml of ethyl acetate and added to the aqueous slurry.The mixture was hydrogenated on a Paar apparatus at 50 p.s.i. at 22° for4 hours. The slurry was filtered through a thin "Celite" pad on asintered glass funnel, the pad was washed with 2×50 ml H₂ O and theaqueous layer of the combined filtrate and washings was separated. Theaqueous layer was washed with 200 ml of diethyl ether, then was cooledto 5° and, with stirring, a solution of 12 g. (0.076 mole) of KMnO₄ in200 ml of H₂ O was added dropwise over a 1/2 hour period, keeping the pHbetween 7.5 and 8.0 by the addition of 40% H₃ PO₄. When the pink colorpersisted for 5 minutes, no more KMnO₄ solution was added. The reactionmixture was stirred with a small amount (approx. 50 mg) of sodiumbisulfite for 1/2 hour, and then the slurry was filtered through a"Celite" pad. The pad was washed with 2×50 ml of H₂ O. The combinedfiltrate and washings were layered with 500 ml of ethyl acetate and,with stirring, the pH was adjusted to 1.5 by the addition of 2 N HCl.The layers were separated and the aqueous layer was saturated withsodium sulfate. It was reextracted with 2×400 ml of ethyl acetate andthe combined ethyl acetate extracts were dried over sodium sulfate for1/2 hour at 5°. The sodium sulfate was removed by filtration and thefiltrate evaporated under reduced pressure to a residue. That residuewas dissolved in 160 ml of acetone and 160 ml of diethyl ether and 50%by weight of potassium 2-ethylhexanoate in n-butanol was added until thesolution was neutral to moist pH paper. The potassium salt of BL-P2013crystallized out, was collected by filtration, washed with diethyl etherand dried. Yield 16 g potassium2β-chloromethyl-2-methylpenam-3-carboxylate sulfone (BL-P2013) (76%),m.p. 202°. The ir and nmr spectra were consistent for the desiredstructure.

Anal. Calcd. for C₈ H₉ ClKNO₅ S: C, 31.42; H, 2.97; N, 4.58. Found: C,31.18; H, 2.98; N, 4.51; H₂ O, 0.93.

EXAMPLE 16 Pivaloyloxymethyl 2β-Chloromethyl-2-methylpenam-3-carboxylateSulfone (BL-P2024) ##STR49##

To a stirred suspension of 14.6 g (0.0487 mole) of potassium2β-chloromethyl-2-methylpenam-3-carboxylate sulfone (BL-P2013) in 200 mlof acetone was added 4 ml of a 10% aqueous solution of sodium iodide andthe mixture was brought to reflux on the steam bath. To this refluxingsuspension was added 14.8 ml. (0.1 mole) of redistilled chloromethylpivalate (bp 34° C. at 7 mm Hg) all at once. The mixture was stirred atreflux for three hours and then cooled to room temperature (22° C.). Thecrystalline solids were collected by filtration, washed with 3×30 ml ofacetone and the combined filtrates were evaporated to an oil underreduced pressure at <22° C. The oil was then taken up in 500 ml of ethylacetate and washed once with water (200 ml) and once with saturated Na₂SO₄ solution (200 ml). The solution was then dried briefly over Na₂ SO₄while being stirred with 2 g of decolorizing carbon with cooling (icebath). After 20 min. the mixture was filtered through a "Celite" pad andthe pad washed with 4×100 ml of ethyl acetate. The combined filtrateswere concentrated under reduced pressure at 22° C. to an oil. The oilwas then further concentrated at about 22° C. and <1 mm Hg to removemost of the residual chloromethyl pivalate. The remaining oil was thentriturated twice with 50 ml portions of n-pentane and then left over theweekend at about 10° C. under n-pentane. The resulting solid crystallinemass was then broken up to a powder under 40 ml of a 4:1 mixture ofdiethyl ether-n-pentane. The product was then collected by filtration,washed with diethyl ether-n-pentane (1:1) then n-pentane and air dried.After drying under high vacuum for four hours over P₂ O₅ there wasobtained 13.37 g pivaloyloxymethyl2β-chloromethyl-2-methylpenam-3-carboxylate sulfone (BL-P2024) about(75%), m.p. 93°-95° C.

Purification of BL-P2024

Approximately 3 g. of crude BL-P2024 (obtained as described above) wasdissolved in 5 ml. of ethyl acetate, placed on a 4.5×40 cm. column ofsilica gel (Mallinckrodt CC-7), and eluted with 4:1 v/v CH₂ Cl₂ -ethylacetate. The fractions containing a single spot at R_(f) 0.84 (TLC onsilica gel plates with 4:1 CH₂ Cl₂ -ethyl acetate, I₂ detection) werecombined and concentrated under reduced pressure to 1.38 g. of acrystalline solid. A portion of this material (900 mg.) was dissolved in5 ml. of ethyl acetate; the resulting solution was filtered, dilutedalmost to the cloud point with petroleum ether ("Skellysolve B") andthen stored at room temperature for three days. The crystals whichformed were collected by filtration, washed with petroleum ether anddried to give 560 mg., m.p. 100°-101°, of purified BL-P2024.

Anal. Calcd. for C₁₄ H₂₀ ClNO₇ S: C, 44.03; H, 5.27; N, 3.67. Found: C,44.11; H, 5.08; N, 3.85.

All tempertures in this application are given in degrees Centigrade.

EXAMPLE 17 Preparation of BL-P2013 Ammonium Salt

1. The free acid of BL-P2013 (250 mg.) dissolved in 20 ml. ofacetone-methanol (1:1 by volume) was filtered to get a clear solution.

2. Anhydrous ammonium solution was prepared by adding 1 ml. of ammoniumhydroxide (30%, reagent grade) to 10 ml. of acetone-methanol (1:1 byvolume) solvent and then 1 gm. of anhydrous magnesium sulfate was addedto that solution with mild agitation and the mixture was filteredthrough a filter paper; the filtrate was designated "anhydrous ammoniumsolution."

3. To the filtrate of Procedure 1, approximately 2 ml. of "anhydrousammonium solution" was gradually added and mixed well.

4. A 100 ml. portion of diethyl ether was mixed with the mixture fromProcedure 3 to precipitate the ammonium salt of BL-P2013.

5. The White ammonium salt was isolated from the solvent and washed with2 portions of 50 ml. each of diethyl ether.

6. The isolated powder was dried at 35° C. vacuum oven for overnight.

7. Analytical data were as follows: Calculated % C 33.7; H 4.6; N 9.8;Found C 33.66; H 4.63; N 10.12; dry by KF Microscopic Examination:crystalline substance.

EXAMPLE 18 Preparation of Non-hygroscopic Sodium Salt of BL-P2013

1. Dissolve 50 mg. of the free acid of BL-P2013 in 4 ml. ofacetone-methanol (1:1 by volume) mixture. Filter to get a clearsolution.

2. Prepare sodium 2-ethylhexanoate solution by dissolving 40 mg. ofsodium 2-ethylhexanoate in 10 ml. of acetone-methanol (1:1 by volume)mixture.

3. To the filtrate of Procedure 1, add the 10 ml. solution of Procedure2 and mix well.

4. A 10 ml. portion of diethyl ether was mixed with the mixture fromProcedure 3 to precipitate the sodium salt of BL-P2013.

5. The white salt was immersed in the diethyl ether for 1-2 hours andthen was isolated from the solvent and washed with 3 portions of 5 ml.each of diethyl ether.

6. The isolated powder was dried at 30° C. vacuum oven for overnight.

EXAMPLE 19 Recrystallization of BL-P2013 ##STR50##

BL-P2013 (400 mg.) was dissolved in a minimum amount of acetone-H₂ O(1:1) by volume and diluted with 10 ml. of acetone, filtered, thendiluted with acetone to about 25 ml., scratched, and after 30 minutesthe crystalline hydrate was collected by filtration, washed well withacetone, air dried and then vacuum dried at <1 mm. Hg overnight.

Yield 280 mg.

Anal. Calcd. for C₈ H₉ ClNOSK.H₂ O: C, 29.67; H, 3.39; N, 4.63; Cl,10.94; H₂ O, 5.55. Found: C, 29.32; H, 3.32; N, 4.44; Cl, 11.31; H₂ O,5.90.

EXAMPLE 20 N,N'-Dibenzylethylenediamine Salt of BL-P2013

BL-P2013+1/2 N,N'-Dibenzylethylenediamine diacetate ##STR51##

306 mg. (0.001 ml) of BL-P2013 was dissolved in 7 ml. H₂ O and added toa solution of 180 mg. (0.0005 mol) of N,N'-dibenzylethylenediaminediacetate in 7 ml. H₂ O. The mixture was stirred and the saltcrystallized and after stirring approximately 10-15 minutes the salt wascollected by filtration and air dried to yieldN,N'-dibenzylethylenediamine salt of BL-P2013 (300 mg). The material wasrecrystallized by dissolving it in approximately 10 ml. of boilingacetone and diluting with ether to the cloud point. 260 mg. of air driedand vacuum dried material was obtained.

Anal. Calcd: C, 51.69; H, 5.42; N, 7.53; Cl, 9.55. Found: C, 49.39; H,5.49; N, 7.05; Cl, 8.96; H₂ O, 1.23 (KF).

EXAMPLE 21 Chloromethyl Ester of BL-P2013 ##STR52##

To a vigorously stirred mixture of 15.25 g (0.05 mol) of BL-P2013 (5),15 g. (0.15 mol) KHCO₃ and 1.7 g (0.005 mol) of tetrabutylammoniumhydrogen sulfate (Aldrich Chem. Co.) in a mixture of 50 ml. water and 50ml. CH₂ Cl₂ there was added dropwise a solution of 9.5 g (0.0575 mol) ofClCH₂ -O-SO₂ Cl in 40 ml. CH₂ Cl₂. The temperature rose to 26° C. andafter the addition (which took about 15 minutes) the mixture was stirredanother 30 minutes. Because the product crystallized out more CH₂ Cl₂(about 400 ml.) was added to obtain a solution. The separated CH₂ Cl₂layer and a 50 ml. CH₂ Cl₂ wash were combined, dried over MgSO₄ withstirring and 2 g of decolorizing carbon ("Darco KB") was added. Afterabout 30 minutes the mixture was filtered, concentrated to about 50 ml.and isopropyl alcohol (150 ml.) was added. The rest of the CH₂ Cl₂ wasthen removed under reduced pressure. The resulting crystallineprecipitate was collected by filtration, washed well with isopropylalcohol and air-dried. After vacuum drying at less than 1 mm. Hg therewas obtained 8.5 g of chloromethyl2β-chloromethyl-2-methylpenam-3-carboxylate sulfone (7). M.p. 116°(dec., darkens above 100° C.).

Anal. Calcd. for C₉ H₁₁ Cl₂ NO₅ S: C, 34.18; H, 3.51; N, 4.43; Cl,22.43. Found: C, 34.16; H, 3.45; N, 4.47; Cl, 22.46; H₂ O, 0.33 (KF).

Estimated purity in the 90-95% range.

Iodomethyl Ester of BL-P2013 ##STR53##

To a stirred mixture of 5 g. (0.0159 mol) of the chloromethyl ester ofBL-P2013 (7) in 25 ml. acetone was added 3 g. (0.02 mol) of sodiumiodide. The resulting slurry was stirred for 17 hours and then cooled toabout 0° C. Two drops of saturated aqueous KHCO₃ were added and themixture was slowly diluted dropwise with water over ten minutes until 50ml. had been added. The slurry underwent a sudden color change fromyellow to grey to purple to black and therefore the crystals wereimmediately collected by filtration and washed with cold acetone-water(1:2) and then isopropyl alcohol (3×10 ml), then diethyl ether andfinally n-pentane and air-dried to yield 5.55 g. (91% yield) of theiodomethyl ester of BL-P2013 (8). M.p. 118°-119° C. with decomposition.Purity estimated at about 90%.

6-[(R)-2-Amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carbonyloxymethyl2β-chloromethyl-2-α-methylpenam-3α-carboxylate sulfone (11) ##STR54##

To a stirred mixture cooled in an ice-bath of 5.46 g. (0.01 mol) of theindicated Dane salt of ampicillin 9 (which was solvated with onemolecule of isopropyl alcohol) in 60 ml. acetone there was added 4.08 g.(0.01 ml) of the iodomethyl ester of BL-P2013 (8) and the resultingnearly clear solution was stirred for five hours with the ice-bathremoved after 30 minutes. Then most of the acetone was removed in vacuumon the roto-vap and the resulting concentrated solution was dissolved in200 ml. cold ethyl acetate which was then washed with 2×50 ml. ice coldwater and 2×100 ml. saturated aqueous Na₂ SO₄. The ethyl acetatesolution was then dried over Na₂ SO₄, filtered and most of the ethylacetate was removed in vacuo on the roto-vap. The residue was trituratedwith 2×200 ml. dry diethyl ether and the resulting solids were collectedby filtration to give 5.5 g. of 10 as a pinkish powder. This powder wasstirred in a mixture of 50 ml. water, 50 ml. n-butanol and 20 ml. ethylacetate while 6 N HCl was added dropwise to pH 2.5. Then occasionally adrop or two of HCl was added to keep the pH at 2.2-2.5 over 45 minutes.When the pH no longer drifted upward there was added to this mixture 100ml. diethyl ether with good stirring. The aqueous phase was separatedand combined with a second 25 ml. H₂ O extract of the organic layer. Theaqueous solution was extracted once with 50 ml. diethyl ether and theether was discarded.

The aqueous layer was then stirred vigorously under a layer of 100 ml.2-butanone (methyl ethyl ketone) while Na₂ SO₄ was added to saturate theaqueous layer. The 2-butanone layer was separated, dried over Na₂ SO₄for 30 minutes in an ice-bath, filtered and concentrated in vacuo tonear dryness. The residual oil was triturated to a solid with n-butanol,washed well with ether, then n-pentane, air-dried and then vacuum-driedover P₂ O₅ at <1 mm Hg pressure to yield 1.6 g.6-[(R)-2-amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carbonylmethyl2β-chloromethyl-2-α-methylpenam-3α-carboxylate sulfone (11) in crudeform. The IR and nmr spectra were consistent with structure 11 but notwith high purity. This solid product was estimated to contain at least40% and perhaps as much as 80%6-[(R)-2-amino-2-phenylacetamido]3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carbonyloxymethyl2β-chloromethyl-2-α-methylpenam-3α-carboxylate sulfone.

EXAMPLE 22 Improved Synthesis of BL-P2013

This procedure simplifies production of BL-P2013 by eliminating theprevious use of catalytic reduction. ##STR55## (See page 633 ofCephalosporins and Penicillins, edited by Edwin H. Flynn, AcademicPress, New York, 1972)

6α-Bromopenicillanic acid sulfoxide (1) (30 g., 0.1 mol) was dissolvedin 1 l. dry CH₂ Cl₂ followed by the addition of 16.2 ml. (0.2 mol)pyridine and 29.8 g. (0.2 mol) trichloroethanol. Then 20 g. (0.1 mol) ofdicyclohexylcarbodiimide was added and the mixture was stirred at 22°for 16 hours. Dicyclohexylurea began to precipitate out; at the end itwas removed by filtration. The filtrate was washed with 200 ml. of 5%aqueous sodium bicarbonate, 200 ml. of 10% phosphoric acid and 100 ml.of saturated aqueous sodium sulfate. The organic phase was dried oversodium sulfate at 5° C. for 30 minutes, filtered and evaporated to anoil. Diethyl ether was added and, with scratching, the product 2crystallized out (27 g., 63% yield). ##STR56##

Compound 2 (26.5 g., 0.062 mol) was dissolved in 500 ml. p-dioxane andthere was added 8.5 ml. (0.078 mole) benzoyl chloride and 8.75 ml.(0.078 mole) quinoline. The solution was refluxed for four hours andthen poured into 1100 ml. water and the product 3 was extracted into2×400 ml. ethyl acetate. The ethyl acetate extracts were combined,successively washed with 200 ml. 5% aqueous sodium bicarbonate, 200 ml.5% phosphoric acid and 200 ml. saturated aqueous sodium sulfate, driedover sodium sulfate at 5° C., for thirty minutes and evaporated to anoil (3) which was used "as is" for the next reaction. ##STR57##

Compound 3 obtained in the previous step was dissolved in 1 l. glacialacetic acid and, with stirring at 22° C., a saturated aqueous solutionof KMnO₄ was added dropwise until a pink color persisted (that is, adrop placed on a piece of filter paper gave a pink coloration). Thenwith cooling 30% H₂ O₂ was added dropwise until a clear solution wasobtained; some white precipitate was present. The solution was pouredinto 2.5 l. water and the product 4 was extracted into 3×500 ml. ethylacetate. The ethyl acetate was washed with 5% aqueous sodium bicarbonateuntil neutral (that is, no more bubbling upon addition), dried oversodium sulfate and evaporated to leave 4 as the residue. It was left at10° C. for one day and then triturated with "Skellysolve B" to yield 9.1g. solid 4. The yield was 28% of theory for steps 2 and 3 combined.##STR58## (See U.S. Pat. No. 4,164,497).

Zinc dust (3.75 g.) was slurried in 5 ml. glacial acetic acid and cooledto 5° C. To this mixture there was added a solution of 4 (3 g.; 0.0057mole) in 15 ml. dimethylformamide and the resulting slurry was stirredat 5° for 2.5 hours.

The zinc was then filtered off and the pale yellow solution was pouredinto 80 ml. of 5% aqueous hydrochloric acid. That mixture was extractedwith 3×25 ml. ethyl acetate. The combined ethyl acetate extracts wereextracted with 3×20 ml. of 5% aqueous sodium bicarbonate, saving theethyl acetate phase after separation.

The bicarbonate extracts were combined, placed under a layer of ethylacetate, adjusted to pH 1.5 by the addition of 2 N HCl and saturatedwith sodium sulfate. The ethyl acetate was separated and the aqueousphase was extracted with 2×30 ml. ethyl acetate.

All of the ethyl acetate phases above were combined, dried over sodiumsulfate and evaporated to an oil (which was the free acid form ofBL-P2013) which was dissolved in about 20 ml. acetone to which 20 ml.diethyl ether was then added. Then 50% potassium 2-ethylhexanoate (KEH)in dry n-butanol was added to neutrality. The product 5 (BL-P2013)crystallized out. After stirring 0.5 hour at 22° it was collected byfiltration to yield 650 mgm. of 5 (37% yield).

A 50 mgm. sample of 5 was dissolved in 0.5 ml. water and 20 mgm.N,N'-dibenzylethylenediamine (DBED) diacetate was added. The DBED saltof 5 crystallized out, was collected by filtration, washed with waterand dried over P₂ O₅ under vacuum to yield N,N'-dibenzylethylenediamine2β-chloromethyl-2-methylpenam-3-carboxylate sulfone (DBED salt of freeacid 5).

Another sample of 5 (450 mgm.) was dissolved in 3 ml. water to which wasadded a solution of 270 mgm. DBED diacetate in 2 ml. H₂ O. Withscratching the DBED salt of 5 crystallized out (430 mgm.).Recrystallization from about 5 ml. boiling acetone yielded 270 mgm.

EXAMPLE 236-[(R)-2-Amino-2-p-hydroxyphenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carbonyloxymethyl2β-chloromethyl-2-α-methylpenam-3α-carboxylate sulfone having theformula ##STR59## is produced by substituting the corresponding Danesalt of amoxicillin for the ampicillin used in the procedure of Example21. EXAMPLE 24 A. Potassium6-β-[N-(1-Methoxycarbonyl-propen(-2-yl)-D-α-amino-(4-hydroxyphenyl)acetamido]-penicillanate##STR60## Experimental

To a stirred suspension of ground (mortar) K₂ CO₃ (0.55 mol) in DMF (625ml) was added 108 ml (01.0 mol) of methylacetoacetate (EKC) followed by209.5 (0.5 ml) of amoxicillin trihydrate and the mixture was stirred 4hours at 22° C.±2° C. and then 2 hours at 0°-5° C. (ice bath). Themixture was filtered* and ether (2.5 liters) was added with goodstirring and the mixture was left to stand for 30 minutes. The ether wasdecanted from the oil and 2×2 liter ether portions (triturates) wereadded and stirred to remove most of the DMF. Then acetone (one liter)was added and with vigorous stirring the product crystallized, wascollected by filtration, washed with acetone, dissolved in DMF (600 ml)and the procedure repeated giving an airdried yield of 144 g ofpotassium6-β-[N-(1-methoxycarbonyl-propen)-2-yl)-D-α-amino-(4-hydroxyphenyl)acetamido]-penicillanate.High-vacuum over P₂ O₅ at <1 mm Hg gave 142 g. Dec. pt. 232°-234° C.

B.2-(β-Chloromethyl)-2-methyl-1,1-dioxypenam-3-carbonyloxymethyl-6-[D-(-)-α-amino-α-phenylacetamido]-penicillanateHydrochloride (BL-P2044) ##STR61## Experimental

To a stirred and cooled solution of 5 g. (0.01 mole) of potassium6-β-[N-(1-methoxycarbonyl-propen(-2-yl)-D-α-amino-(4-hydroxyphenyl)acetamido]-penicillanatein 17 ml of dry DMF (dried over 3A molecular sieves), was added 4 g.(0.01 mole) iodomethyl ester of BL-P2013 in portions over a 15 minuteperiod at 3°-8° C. The resulting solution was stirred an additional 15minutes and then poured onto a vigorously stirred, ice cold mixture of80 ml of ethyl acetate and 40 ml of saturated CaCl₂ solution. Themixture was filtered rapidly with suction through a "Dicalite" pad. Theorganic layer was washed twice more with 30 ml portions of saturatedCaCl₂ solution. The ethyl acetate solution was then concentrated invacuo at 22° C. on the flash evaporator to a volume of about 10 ml then50 ml of n-butanol and 50 ml of H₂ O were added plus 50 ml of ethylacetate and 3 N HCl was added dropwise, with good stirring to maintainthe pH at 2.5. The pH was kept at 2.5±0.5 pH units for 30 minutes thenether was slowly added (about 100-150 ml). The aqueous layer wasseparated and another 25 ml H₂ O extract was taken and combined with thefirst. The H₂ O layer was then layered with methyl ethyl ketone(2-butanone) (MEK) 200 ml and salt (NaCl) added until the mixture wassaturated. Stirring was continued vigorously for about 15 minutes. The"MEK" layer was separated, dried briefly over Na₂ SO₄ at 10° C. (icebath), filtered, then concentrated in vacuo on the roto-vap to neardryness. The residual oil was triturated and stirred with 30 ml ofn-butanol for one hour until a nearly white solid was obtained. Theproduct was collected by filtration, washed with n-butanol then ether(10×20 ml) then n-pentane (10×20 ml) and air dried to yield 800 mg of2-(β-chloromethyl)-2-methyl-1,1-dioxypenam-3-carbonyloxymethyl-6-[D-(-)-α-amino-α-phenylacetamido]-penicillanatehydrochloride. After high-vacuum drying over P₂ O₅ at <1 mm Hg for 18hours there was 760 mg.; and the nmrs run in DMSO and DMSO+D₂ O wereentirely consistent with the desired structure. Dec. pt. 200°-205° C.(darken above 150° C.).

EXAMPLE 25 2β-Chloromethyl-2α-methyl-1,1-dioxopenam-3-carbonyloxymethyl6-(D-2,2-dimethyl-5-oxo-4-phenyl-1-imidazolidinyl)penicillanateHydrochloride (BL-P2048) ##STR62## Experimental

To a stirred, partial solution of 2.2 g (0.051 mol) of potassiumhetacillin in 9 ml of dry DMF (dried over 3A molecular sieves), cooledto -20° C., was added, in portions, 2.2 g (0.0054 mol) of iodomethyl2β-chloromethyl-2α-methyl-1,1-dioxopenam-3-carboxylate (iodomethyl esterof BL-P2013) over a 10 minute period. The ice-salt cooling bath wasremoved and replaced by an ice cooling bath. The solution was stirred atabout 3° C. for 15 minutes then the ice bath was removed and thesolution was allowed to come to room temperature over a 30 minuteperiod. The dark solution was diluted with 250 ml of ice cold ethylacetate and washed with 5×30 ml of ice cold water and once withsaturated aqueous sodium sulfate solution. The ethyl acetate solutionwas then stirred in an ice bath with about 1 g of decolorizing carbon("Darko KB") and anhydrous sodium sulfate for 30 minutes. The mixturewas filtered and the carbon and Na₂ SO₄ washed with 4×30 ml of ethylacetate. The combined filtrates were concentrated under reduced pressureat 22° C. to a frothy solid. This was triturated twice with ether (2×25ml) and then n-pentane (2×25 ml) and finally ether again. The solidoff-white powder was collected by filtration, washed with n-pentane, airdried and vacuum dried over P₂ O₅ at <1 mm. Yield, 2.45 g of crudeester.

This material was dissolved in 35 ml of ethyl acetate and then, withgood stirring, a solution of dry HCl in anhydrous ether was added a fewdrops at a time until the ethyl acetate mixture was pH 2 to moist pHpaper. A small amount of colored solids were filtered off (100-200 mg)and discarded. Ether 200 ml) was added with stirring and the whitesolids that precipitated were collected by filtration, generously washedwith ether and then pentane, air dried then vacuum dried to give 700 mgof product 2β-chloromethyl-2α-methyl-1,1-dioxopenam-3-carbonyloxymethyl6-(D-2,2-dimethyl-5-oxo-4-phenyl-1-imidazolidinyl)penicillanatehydrochloride. The ir and nmr spectra were consistent with the desiredstructure. Dec. pt. >140° C. indefinite.

Anal. Calcd for C₂₈ H₃₃ ClN₄ O₉ S₂.HCl: C, 47.49; H, 4.85; N, 7.98; Cl,10.00. Found: C, 47.25; H, 4.57; N, 8.25; Cl, 9.12; K.F. H₂ O, 0.90.

IR: 1790 cm⁻¹, 1730 cm⁻¹. H-NMR(DMSO).

δ1.3-1.9(m, 15H,(5,CH₃ 's)), 3.1-3.8(m,2H,C₆ --H₂ (CMPASO₂));3.8-4.4(m,2H,--CH₂ Cl) 4.6(s,1H,C₃ H(Heta)); 4.95(s,1H,C₃ H(CMPASO₂));5.25(m,1H,C₅ --H(CMPASO₂)); 5.4-5.6(m,2H,C₅ H+C₆ H(Heta));5.65(s,1H,φ--CH); 6.0(s,2H,--O--CH₂ --O); 7.3-7.9 ##STR63## A secondcrop of 800 mg was obtained by adding excess ether-HCl solution to thefiltrate (which did not include the pentane washes) and immediatelyfiltering off the snow-white ppt. and washing and drying as before.Decomposition point: >150° C. indefinite. The NMR spectrum was betterdefined. IR: 1790 cm⁻¹ (β-lactams and esters overlapping) 1730 cm⁻¹imidazolidine carbonyl. H-NMR(DMSO) (Varian XL-100): δ1.3-1.9(m,15H,5,CH₃ 's); 3.1-3.8(m,2H,C₆ --H₂ (CMPASO₂)); 3.8-4.4(m,2H,--CH₂Cl); 4.6 (s,1H,C₃ H(Heta)); 4.95(s,1H,C₃ -H(CMPASO₂)); 5.25(m,1H,C₅-H(CMPASO₂)); 5.4-5.6(m,2H,C₅ H,C₆ H, Heta), 5.65(s,1H,φ--CH--);6.0(s,2H,O--CH₂ --O); 7.3-7.9 ##STR64##

Anal. Calcd. for C₂₈ H₃₃ ClN₄ O₉ S₂.HCl: C, 47.49; H, 4.85; N, 7.98; Cl,10.00. Found: C, 47.44; H, 4.80; N, 8.11; Cl, 9.69; K.F. H₂ O, 1.25.

EXAMPLE 26 A. Potassium Heta-amoxicillin ##STR65## Experimental

To a stirred suspension of 40.4 g (0.1 mole) of BL-P1415 in 400 ml ofacetone was added 38.5 ml (0.1 ml) of "KEH" (potassium 2-ethylhexanoatein n-butanol) and the mixture was stirred and scratched. 400 ml moreacetone was then added and the slurry stirred for 2 hours. The productwas collected by filtration, washed with 4×100 ml. acetone, 4×200 ml.ethyl acetate, 4×200 ml ether then 4×100 ml of n-pentane and air driedovernight to give 42 g. High-vacuum drying at <1 mm Hg for 6 hours gave40.5 g of potassium heta-amoxicillin. (92% yield). Dec. pt. 208°-210° C.

B. 2β-Chloromethyl-2α-methyl-1,1-dioxopenam-3-carbonyloxymethyl6-[D-2,2-dimethyl-5-oxo-4-(4-hydroxyphenyl)-1-imidazolidinyl]-penicillanateHydrochloride (BL-P2049) ##STR66##

To a stirred solution of 4.4 g (0.01 mol) of BL-P1415 (K-salt) in 10 mlof dry DMF at 3° C. (ice bath) was added in portions 4 g (0.01 mol) ofthe iodomethyl ester of BL-P2013 over a 10 minute period. Stirring wascontinued for 20 minutes at 3° C. The resulting solution was dilutedwith 100 ml of ice cold ethyl acetate and 20 ml of ice cold water. TheH₂ O layer was separated and the organic layer washed 4 more times withice water (25 ml) then once with aqueous saturated Na₂ SO₄ solution (40ml). The organic layer was separated and filtered through anhydrous Na₂SO₄ and the Na₂ SO₄ washed 3×25 ml of ethyl acetate. The combinedfiltrates were concentrated under reduced pressure to a froth at 22° C.The froth was re-dissolved in 100 ml of ethyl acetate and filtered. Itwas then stirred rapidly while dry HCl dissolved in dry ether was addeddropwise until no more precipitate formed. The precipitate wasimmediately collected by filtration, washed with dry ether then pentane(5×25 ml) and air dried. Yield 3 g. After vacuum drying over P₂ O₅ for 5hours there was obtained 2.9 g of product2β-chloromethyl-2α-methyl-1,1-dioxopenam- 3-carbonyl-oxymethyl6-[D-2,2-dimethyl-5-oxo-4-(4-hydroxyphenyl)-1-imidazolidinyl]-penicillanatehydrochloride. Dec. pt. 188°-195° with slow dec. above 140° C. The irand nmr spectra were consistent with the desired structure.

Anal. Calcd. for C₂₈ H₃₃ ClN₄ O₁₀ S₂.HCl: C, 46.58; H, 4.75; N. 7.76;Cl. 9.82. Found: C, 47.34; H, 5.08; N, 7.77; Cl, 9.26; K.F. H₂ O, 1.07.

IR: 1800 cm⁻¹, 1750 cm⁻¹ (shoulder), 1140 cm⁻¹, 1330 cm⁻¹SO₂.H-NMR(DMSO) (Varian XL-100) δ6.7-7.5 ##STR67## 5.95 (s,2H,--O--CH₂--O--), 5.6(s,1H,φ--CH--), 5.4-5.6 (m,2H,C₅ H,C₆ H(Hetamox)); 5.1-5.3(m,1H,C₅ --H(CMPASO₂)), 5.95 (s,1H,C₃ H(CMPASO₂)), 4.6 (s,1H,C₃H(Hetamox)), 3.8-4.5 (m,2H,--CH₂ Cl), 3.1-3.9 (m2H,C₆ --H₂ (CMPASO₂)),1.1-1.9 (m,15H,5 CH₃ 's).

EXAMPLE 27

The following is an alternate synthesis of BL-P2013 which eliminates theneed for a catalytic hydrogenation. The synthesis is carried out usingthe trichloroethyl ester which is removed at the last step along withthe 6-bromo group by a zinc-acetic acid reduction. ##STR68##

2',2',2'-Trichloroethyl 6α-bromopenicillanate Sulfoxide

To 1 l of dry methylene chloride (dried over Linde 4 A molecular sieves)was added 30 g (0.1 mole) of 6α-bromopenicillanic acid sulfoxidefollowed by 16.2 ml (0.2 mole) pyridine and 29.8 g (0.2 mole) oftrichloroethanol. Then 20 g (0.1 mole) of dicyclohexylcarbodiimide (DCC)was added and the mixture stirred at 22° for 16 hours. The precipitatewas filtered off and the filtrate was washed with 200 ml of 5% aqueoussodium bicarbonate, 200 ml of 10% aqueous phosphoric acid and 100 ml ofsaturated aqueous sodium sulfate. The methylene chloride solution wasdried over anhydrous sodium sulfate at 5° for 1/2 hour, filtered andevaporated to an oil. Ethyl ether was added and with scratching, theproduct crystallized out. It was collected by filtration, washed withethyl ether, and dried to yield 27 g of 2',2',2'-trichloroethyl6α-bromopenicillanate sulfoxide (63% yield), mp 125°-126°. The nmrspectrum was consistent for the desired structure.

Anal. Calcd. for C₁₀ H₁₁ BrCl₃ NO₄ S: C, 28.09; H, 2.59; N, 3.28. Found:C, 29.40; H, 2.97; N, 3.59; K.F. (H₂ O), 0.40.¹

2',2',2'-Trichloroethyl6α-bromo-2β-chloromethyl-2α-methylpenam-3-carboxylate

To 500 ml of 1,4-dioxane was added 26.5 g (0.062 mole) of2',2',2'-trichloroethyl 6α-bromopenicillanate sulfoxide followed by 8.5ml (0.078 mole) of benzoyl chloride and 8.75 ml (0.078 mole) ofquinoline. The solution was refluxed for 4 hours. It was then pouredinto 1100 ml H₂ O and extracted with 2×400 ml ethyl acetate. The ethylacetate extracts were combined, washed with 200 ml 5% aqueous sodiumbicarbonate, 200 ml 5% aqueous phosphoric acid and 200 ml of saturatedaqueous sodium sulfate. The ethyl acetate extracts were dried overanhydrous sodium sulfate at 5° for 1/2 hour, filtered, and the filtrateevaporated to give 2',2',2'-trichloroethyl6α-bromo-2β-chloromethyl-2α-methylpenam-3-carboxylate as an oil. Thismaterial was used for the next reaction without further purification.²

2',2',2'-Trichloroethyl6α-bromo-2β-chloromethyl-2α-methylpenam-3-carboxylate Sulfone

The oil from the previous reaction was dissolved in 1 l of glacialacetic acid and, with stirring at 22°, a saturated aqueous solution ofpotassium permanganate was added dropwise until a pink color persistedfor 5 minutes. (A drop placed on a piece of filter paper giving a pinkcoloration). Then a 30% hydrogen peroxide solution was added dropwiseuntil a clear solution was obtained (some white precipitate waspresent). The solution was poured into 21/2 liters of H₂ O and theproduct extracted into 3×500 ml of ethyl acetate. The combined ethylacetate extracts were washed with 5% aqueous sodium bicarbonate solutionuntil neutral. It was dried over sodium sulfate at 5° for 1/2 hour andevaporated to a residue. It was triturated with heptanes ("SkellysolveB") to yield 9.1 g of solid 2',2',2'-trichloroethyl6α-bromo-2β-chloromethyl-2α-methylpenam-3-carboxylate sulfone. (28%yield for two steps). The nmr spectrum was consistent for structure.³

BL-P2013

A slurry of 3.75 g of zinc dust in 5 ml of glacial acetic acid wascooled to 5° and to it was added a solution of 3 g (0.0057 mole) of2',2',2'-trichloroethyl6α-bromo-2β-chloromethyl-2α-methylpenam-3-carboxylate sulfone in 15 mlof dimethylformamide. The slurry was stirred at 5° for 21/2 hours andthe zinc was removed by filtration. The pale yellow filtrate was pouredinto 80 ml of 5% aqueous hydrochloric acid and extracted with 3×25 ml ofethyl acetate. The combined ethyl acetate extracts were extracted with3×20 ml of 5% aqueous bicarbonate. (The ethyl acetate contained 600 mgof starting material). The bicarbonate extracts were combined and, undera layer of 30 ml of ethyl acetate, the pH was adjusted to 1.5 by theaddition of 2 N HCl. The solution was saturated with sodium sulfate andthe ethyl acetate separated. The aqueous layer was extracted with 2×30ml of ethyl acetate. The combined ethyl acetate extracts were dried oversodium sulfate at 5° for 1/2 hour and evaporated to an oil. The oil wasdissolved in 20 ml of acetone and 20 ml of ether added. Then, 50%potassium 2-ethylhexanoate (KEH) in n-butanol was added dropwise untilthe pH of the solution was neutral and the product crystallized out. Itwas collected by filtration to yield 650 mg. (47% yield) of BL-P2013.The nmr spectrum was consistent for structure.⁴

DBED Salt of BL-P2013

A solution of 270 mg (0.00075 mole) at N,N'-dibenzylethylenediamine(DBED) diacetate in 2 ml H₂ O was added to 450 mg (0.0015 mole) ofBL-P2013 in 3 ml H₂ O. With scratching, the DBED salt crystallized outand was filtered off to yield 430 mg. It was recrystallized bydissolving it in 5 ml of boiling acetone and after filtering, theproduct crystallized out of the cold filtrate. It was left at 5° for 1/2hour, collected by filtration and washed with 5 ml of ethyl ether toyield 270 mg (47% yield) of DBED salt of BL-P2013.

Anal. Calcd. for C₁₆ H₁₉ ClN₂ O₅ S: C, 49.68; H, 4.95; N, 7.24; Cl,9.16. Found: C, 49.63; H, 5.50; N, 6.99; Cl, 8.37; K.F. (H₂ O), 1.32.

The nmr spectrum was consistent for structure.

BL-P2013.K Salt from the DBED Salt

In a mixture of 2 ml of H₂ O and 6 ml of ethyl acetate, 190 mg (0.0005mole) of BL-P2013 DBED salt was suspended and 40% aqueous phosphoricacid was added until the pH was 2. The aqueous layer was separated andreextracted with 3 ml of ethyl acetate. The combined ethyl acetateextracts were dried over sodium sulfate at 5° for 1/2 hour and filtered.The filtrate was evaporated to a residue and redissolved in 2 ml ofacetone and 2 ml of ether. Then 50% potassium 2-ethylhexanoate inn-butanol was added dropwise until the pH was neutral. The potassiumsalt of BL-P2013 crystallized out, was collected by filtration and driedto yield 90 mg (60% yield). The nmr spectrum was consistent forstructure.

Anal. Calcd. for C₈ H₉ ClKNO₅ S.1/2H₂ O: C, 30.52; H, 3.20; N, 4.45; Cl,11.26; KF, 2.86. Found: C, 30.51; H, 3.50; N, 4.38; Cl, 9.98; KF, 3.35.

The CP-45899 content of this material is estimated to be approximately5%.

Footnotes BIOLOGICAL DATA

The product of Example 1, Compound 5, having the structure ##STR69##will be referred to below as BL-P2013.

Although at best a very weak antibacterial agent itself, BL-P2013inhibits β-lactamases and protected ceforanide and amoxicillin fromdestruction by β-lactamase-producing bacteria in vitro and in vivo whenused in combination with those two agents.

                  TABLE 1                                                         ______________________________________                                        Antibacterial Activity of the New Sulfone                                                      MIC (mcg/ml)                                                 Organism           BL-P2013  Ampicillin                                       ______________________________________                                        S. pneumoniae                                                                              A-9585      16      0.004                                        S. pyogenes  A-9604      63      0.004                                        S. aureus    A-9537    >125      0.16                                         S. aureus +  A-9537    >125      0.06                                         50% serum                                                                     S. aureus Pen-                                                                             A-9606    >125      >125                                         Res                                                                           S. aureus Meth-                                                                            A15097    >125      125                                          Res                                                                           S. faecalis  A20688    >125      0.13                                         E. coli      A15119    >125      1                                            E. coli      A20341-1  >125      >125                                         K. pneumoniae                                                                              A15130    >125      125                                          K. pneumoniae                                                                              A20468    >125      >125                                         P. mirabilis A-9900    >125      0.13                                         P. vulgaris  A21559    >125      125                                          P. morganii  A15153    >125      >125                                         P. rettgeri  A21203    >125      4                                            S. marcescens                                                                              A20019    >125      16                                           E. cloacae   A-9659    >125      63                                           E. cloacae   A-9656    >125      >125                                         P. aeruginosa                                                                              A-9843A   >125      >125                                         P. aeruginosa                                                                              A21213    >125      >125                                         ______________________________________                                    

                                      TABLE 2                                     __________________________________________________________________________    Anti-Bacteroides Activity of Ceforanide and Amoxicillin                       Alone and in Combination with BL-P2013                                                  MIC (mcg/ml)*                                                             Beta-   Ceforanide Amoxicillin                                                lact-                                                                             Cefo-                                                                             + BL-P2013                                                                           BL-P                                                                              + BL-P2013                                                                           Amoxi-                                        Organism                                                                            amase                                                                             ranide                                                                            (1:1)  2013                                                                              (1:1)  cillin                                        __________________________________________________________________________    B. fragilis                                                                   A21916                                                                              +   63   -2    >125                                                                               -2    8                                             A22053                                                                              +   32   -4    63   -2    8                                             A22021                                                                              +   32   -2    32    2    4                                             A21875                                                                              +   32   -4    63   -2    8                                             A22534                                                                              +   >125                                                                               --32  125  --16  >125                                          A22967                                                                              +   63   -8    63   -2    8                                             A22693                                                                              +   63   -4    63   -2    16                                            A22694                                                                              +   125  --16  63    -2   16                                            A22695                                                                              +   >125                                                                                16   32   -4    125                                           A22696                                                                              +   >125                                                                                32   63   -8    >125                                          A22533                                                                              +   >125                                                                                32   32  32     >125                                          A22535                                                                              +   >125                                                                               --32  125  --32  >125                                          A22792                                                                              +   >125                                                                               -8    32   -4    125                                           A22793                                                                              +   32   -4    32   -2    8                                             A22794                                                                              +   32   -4    32   -2    8                                             A22795                                                                              +   63   -4    32   -4    16                                            A22797                                                                              +   63   -4    63   -2    16                                            A22798                                                                              +   32   -4    63   -2    8                                             B. theta                                                                      iotaomicron                                                                   A22277                                                                              +   125  -4    63   -2    16                                            A22279                                                                              +   125  -8    63   -4    16                                            Bacteroides                                                                   species                                                                       A20934                                                                              +   32   -4    32   -2    8                                             A21959                                                                              +   63   -4    32   -2    16                                            A20929                                                                              +   63   -4    32   -2    16                                            A21954                                                                              +   63   --16  63   -2    16                                            A20933                                                                              +   63   --16  63    4    8                                             A20930                                                                              +   125  -8    125  -4    32                                            A20931                                                                              +   63   -4    32   -2    16                                            A20927-1                                                                            -   0.5 1      63    0.13 0.13                                          A20935                                                                              -   2   2      125   0.13 0.13                                          __________________________________________________________________________     -- good synergism                                                               marginal synergism                                                          *Minimum inhibitory concentration (MIC) determined by the agar dilution       method using 50X dilutions of 24 hour cultures as inocula dispensed bu th     Steer's inoculator. Assay medium composed of Brucella Agar plus 5% laked      sheep blood and 10 mcg/ml vitamin K.                                     

                  TABLE 3                                                         ______________________________________                                        Therapeutic Efficacy of Amoxicillin in Combination with                       BL-P2013 in Mice Experimentally Infected with a Beta-                         Lactamase Strain of Staphylococcus aureus                                     Challenge   PD.sub.50 /Treatment (mg/kg)                                      Organ-                                                                              (No. of   Amoxicillin (A)                                                                           BL-P2013 (B)                                                                           A+B (1:1)                                ism   (Organisms)                                                                             IM      PO    IM   PO    IM   PO                              ______________________________________                                        S.    5 × 10.sup.8                                                                      >800    >800  >50  >200  6.3  44                              aureus                                                                              5 × 10.sup.8                                                                      >800    >800  >50  >200  19   77                              A-9606                                                                              7 × 10.sup.8                                                                      >800    --    >50  --    9.6  --                              ______________________________________                                         Treatment schedule: Drugs administered 0 and 2 hours postinfection.      

                                      TABLE 4                                     __________________________________________________________________________    Blood levels of BL-P2013 and its Pivaloyloxymethyl Ester                      (BL-P2024) After Oral Adminstration to Mice                                              Blood Level (μg/ml)                                                                         Half-                                             Dose       Minutes After Adminstration                                                                    life Assay                                        Compound                                                                            (mg/kg)                                                                            15 30 60                                                                              90 120                                                                              150                                                                              (min.)                                                                             Organism                                     __________________________________________________________________________    BL-P2013                                                                            100  4.5                                                                              5.1                                                                              3.9                                                                             2.3                                                                              1.5                                                                              0.8                                                                              50   E. coli                                                                       A-9675                                             100  4.6                                                                              4.1                                                                              3 <2.6                                                                             <2.6                                                                             <2.6                                                                             --   S. aureus                                                                     A-9606                                             200  7.4                                                                              9.8                                                                              7.1                                                                             4.1                                                                              2.8                                                                              <2.6                                                                             50   S. aureus                                                                     A-9606                                       BL-P2024                                                                            100  12.8                                                                             12.9                                                                             9.7                                                                             7.2                                                                              5.5                                                                              4.2                                                                              70   E. coli                                                                       A-9675                                             100  13.1                                                                             12 8 5.7                                                                              3.8                                                                              <2.5                                                                             60   S. aureus                                                                     A-9606                                             200  14.7                                                                             14.4                                                                             9.8                                                                             8.7                                                                              5.1                                                                              <2.5                                                                             60   S. aureus                                                                     A-9606                                       __________________________________________________________________________     Values represent averages of two to four tests.                          

                  TABLE 5                                                         ______________________________________                                        Blood Levels and Half-Life of BL-P2024 After Oral                             Administration of Various Doses to Mice                                                    Blood Level (μg/ml)                                           Dose         Minutes After Administration                                                                      t1/2                                         Compoound                                                                             (mg/kg)  15     30   60  90   120  150 (min.)                         ______________________________________                                        BL-P2024                                                                               25      5.9    6.2  3.6 1.9  1.3  0.7 40                                      50      7.7    9.5  6.3 4.7  3.5  2.3 60                                     100      12.3   12.2 9.4 7.2  5.7  4.6 85                                     200      14.7   14.4 9.8 8.7  5.1  <2.5                                                                              60                             ______________________________________                                         BL-P2024 was suspended in TweenCMC-water.                                     Values are averages of 2 experiments for 25 and 50 mg/kg doses, 5 to 6        experiments for the 100 mg/kg dose, and 2 to 3 experiments for the 200        mg/kg dose.                                                                   Assay organism: E. coli A9675 for all doses except for 200 mg/kg of           BLP2024 (S. aureus A9606).                                               

                                      TABLE 6                                     __________________________________________________________________________    Therapeutic Efficacy of Amoxicillin in Combination with BL-P2013 in Mice      Experimentally Infected with β-Lactamase Producing Strains of S.         aureus and E. coli                                                            Challenge  PD.sub.50 /Treatment (mg/kg)                                       (No. of    Amoxicillin:BL-P2013      Bl-P                                     Organism                                                                           Organisms)                                                                          Amoxicillin                                                                       4:1   2:1 1:1 1:2 1:4 2013                                     __________________________________________________________________________    S. aureus                                                                          7 × 10.sup.8                                                                  >200                                                                              >200:>50                                                                            44:22                                                                             33:33                                                                             38:76                                                                              25:100                                                                           >200                                     A9606                                                                              2 × 10.sup.9                                                                  >800      33:17                                                                             14:14                                                                             10:20   >200                                     S. aureus                                                                          4 × 10.sup.8                                                                  >200                                                                              132:66                                                                              78:39                                                                             44:44       >200                                     A15091                                                                             5 × 10.sup.8                                                                  >800      114:57                                                                            100:100                                                                            66:132 >200                                     S. aureus                                                                          6 × 10.sup.8                                                                  >200                                                                              174:44                                                                              44:22                                                                             25:25                                                                             22:44                                                                             19:76                                                                              174                                     A20379                                                                             5 × 10.sup.8                                                                  >200      50:25                                                                             43:43                                                                             35:70   >200                                     E. coli                                                                            5 × 10.sup.5                                                                  >200                                                                               25:6.3                                                                              11:5.5                                                                           11:11       >200                                     A20649                                                                             6 ×10.sup.5                                                                   >200                                                                              20:5  8:4 6:6 10:20                                                                             63:25                                                                             >200                                     E. coli                                                                            7 × 10.sup.5                                                                  >200                                                                                6:1.5                                                                             6:3 5:5         >200                                     A21223                                                                             6 × 10.sup.5                                                                  >200                                                                              8:2   6:3 6:6 4:8 3.5:14                                                                            >200                                          7 × 10.sup.5                                                                  >200        7:3.5         >200                                     E. coli                                                                            8 × 10.sup.5                                                                  >200                                                                              8:2   6:3 3:3 4:8  4:16                                                                             >200                                     A9675                                                                              7 × 10.sup.5                                                                  >100                                                                               10:2.5                                                                             6:3 7:7 2.5:5   >100                                          6 × 10.sup.5                                                                  >400                                                                               13:3.3                                                                             6:3 5:5  5:10   >200                                     __________________________________________________________________________     Treatment schedule: Drugs were administered orally at 0 and 2 hours           postinfection.                                                                Drugs were suspended in TCMC.                                            

                                      TABLE 7                                     __________________________________________________________________________    Therapeutic Efficacy of Amoxicillin in Various Combinations with the          Pivaloyloxymethyl Ester (BL-P2024) of BL-P2013 in Mice Experimentally         Infected                                                                      with β-Lactamase Producing Strains of S. aureus and E. coli              Challenge        PD.sub.50 /Treatment (mg/kg).sup.a                           (No. of    Drug- Amoxi-                                                                            Amoxicillin:BL-P2024                                                                        BL-P                                       Organism                                                                           Organisms)                                                                          Vehicle.sup.b                                                                       cillin                                                                            4:1  2:1  1:1 2024                                       __________________________________________________________________________    S. aureus                                                                          5 × 10.sup.8                                                                  TCMC  >800     66:33                                                                              57:57                                                                             >200                                       A15091                                                                        S. aureus                                                                          5 × 10.sup.8                                                                  TCMC  >800     12:6 9:9  200                                       A20379                                                                        E. coli                                                                            9 × 10.sup.5                                                                  50%   >100                                                                              12:12                                                                              6:3  3:3  >25                                       A9675      DMSO                                                               E. coli                                                                            7 × 10.sup.5                                                                  50%   >100                                                                               10:2.5                                                                              7:3.5                                                                            4:4  >25                                       A9675      DMSO                                                               E. coli                                                                            7 × 10.sup.5                                                                  TCMC  >100                                                                              8:2  6:3  5:5  >25                                       A9675                                                                         E. coli                                                                            6 × 10.sup.5                                                                  TCMC  >400                                                                              9.2:2.4                                                                              7:3.5                                                                            7:7 >200                                       A9675                                                                         __________________________________________________________________________     .sup.a Mice treated orally at 0 and 2 hours postinfection.                    .sup.b Amoxicillin:BLP2024 combinations were soluble in 50% DMSO.             Amoxicillin was soluble in TCMC (aqueous Tweencarboxymethylcellulose)         whereas BLP2024 was administered as a suspension in that vehicle.        

                                      TABLE 8                                     __________________________________________________________________________    Oral Mouse Blood Levels of BL-P2036 and BL-P2013 or BL-P2024                             Blood Levels (mcg/ml)                                              Dose       Minutes After Administration                                       Compound                                                                            (mg/kg)                                                                            15    30    60    90   120  150                                    __________________________________________________________________________    Test Number                                                                   BL-P2036                                                                            25   1.7   2.3   0.9   <0.6 <0.6 <0.6                                              (1.3-2.3)                                                                             (2-2.6)                                                                           (0.6-1.3)                                              BL-P2036                                                                            50   2.8   3.3   2.5   1.3  0.8  <0.6                                              (1.6-4.8)                                                                             (2-5.3)                                                                           (1.2-5.3)                                                                           (0.7-2.4)                                                                          (0.5-1.5)                                   BL-P2036                                                                            100  3.6   3.9   3.2   2    1.5  0.8                                               (2.6-4.9)                                                                           (2.9-5.1)                                                                           (2.4-4.4)                                                                           (1.5-2.7)                                                                          (1.1-2.1)                                                                          (0.7-1.0)                              BL-P2013                                                                            25   1.3   1.4   1.0   <0.6 <0.6 <0.6                                              (1.1-1.6)                                                                           (1.2-1.6)                                                                           (0.8-1.2)                                              BL-P2013                                                                            50   2.2   2.6   1.9   1.5  1.1  0.8                                               (1.8-2.7)                                                                           (2.1-3.2)                                                                           (1.4-2.6)                                                                           (1.1-2.1)                                                                          (0.8-1.4)                                                                          (0.5-1.3)                              BL-P2013                                                                            100  3.4   4.5   3.9   3.6  2.7  2.1                                               (2.1-5.4)                                                                           (3.6-5.7)                                                                           (3.4-4.6)                                                                           (2.5-5)                                                                            (1.7-4.3)                                                                          (1.4-3.2)                              __________________________________________________________________________     Compounds prepared in TweenCMC-H.sub.2 O                                      Values in parentheses are 95% confidence limits.                              Assay Organism:E. coli A9675 (pH = 6.6, 0.1% inoculum)                   

    Test Number 2                                                                 BL-P2024                                                                            25   3.7   4.3   2.3   0.8  <0.6 >0.6                                              (2.6-5.5)                                                                             (3-6.2)                                                                           (0.9-5.8)                                                                           (0.4-1.8)                                        BL-P2024                                                                            50   5     4.5   4.9   3.6  2.6  1.7                                                (0.8-33.3)                                                                          (1.5-13.4)                                                                         (2.4-9.8)                                                                           (3.1-4.1)                                                                          (1.5-4.3)                                                                          (0.9-3.1)                              BL-P2024                                                                            100  8.3   9.5   7.8   4.8  3.9  3.7                                                (5.5-12.6)                                                                          (5.2-17.5)                                                                          (4.2-14.3)                                                                         (3.4-6.7)                                                                          (2.2-6.8)                                                                          (2.8-5)                                BL-P2036                                                                            25   3.6   3.3   2.1   1.4  0.8  >0.6                                              (2.2-5.9)                                                                           (2.2-4.7)                                                                           (1.6-2.8)                                                                           (0.7-2.8)                                                                          (0.4-1.7)                                   BL-P2036                                                                            50   4.0   3.6   2.2   2.1  1.5  1.0                                                 (3-5.3)                                                                           (1.4-9)                                                                             (1.4-3.7)                                                                           (1.3-3.6)                                                                          (0.6-3.6)                                                                          (0.5-2.4)                              BL-P2036                                                                            100  4.9   6.5   4.5   2.8  1.9  1.3                                               (3.7-6.5)                                                                            (3.7-11.5)                                                                         (3.4-5.9)                                                                           (2.2-3.6)                                                                            (1-3.7)                                                                          (0.4-4)                                __________________________________________________________________________     Compounds prepared in 5% Propylene Glycol & TweenCMC-H.sub.2 O                Values in parentheses are 95% confidence limits.                              Assay Organism: E. Coli A9675 (pH = 6.6, 0.1% inoculum)                  

    Test Number 3                                                                 BL-P2013                                                                            25   1.4   2.0   1.4   1.2  <1.2 <1.2                                              (1.1-1.9)                                                                           (1.5-2.6)                                                                           (0.9-2.1)                                                                           (0.6-2.3)                                        BL-P2013                                                                            50   4.6   5.5   3.6   3.1  2    1.1                                                 (3-6.9)                                                                             (3-10.1)                                                                            (2-6.4)                                                                           (1.7-5.7)                                                                          (0.8-5)                                                                            (0.8-1.5)                              BL-P2013                                                                            100  6.8   11.5  6.4   4.2  3.3  2.5                                               (4.8-9.6)                                                                           (7.3-18)                                                                             (3.2-12.8)                                                                         (2.1-8.6)                                                                          (1.4-7.6)                                                                          (0.7-8.9)                              BL-P2036                                                                            25   4.3   4.5   3.5   2.5  2.2  2.0                                               (2.9-6.5)                                                                           (2.2-9.2)                                                                           (1.7-7.2)                                                                           (1.3-4.8)                                                                            (1-4.9)                                                                            (1-3.7)                              BL-P2036                                                                            50   4.6   4.8   3.5   2.2  1.9  1.8                                               (2.3-9.2)                                                                           (3.3-7.1)                                                                           (1.8-7)                                                                             (1.2-4)                                                                            (1.2-3)                                                                            (0.3-4.6)                              BL-P2036                                                                            100  7.1   6.7   5.8   4.3  4    2.7                                                (3.1-16.2)                                                                          (3.6-12.3)                                                                         (3.9-8.5)                                                                           (3-6)                                                                              (2.8-5.6)                                                                          (1.8-4)                                __________________________________________________________________________      Compounds prepared in 5% Propylene Glycol & TweenCMC-H.sub.2 O.              Values in parentheses are 95% confidence limits.                              Assay Organism: E. coli A9675 (pH = 6.6, 0.1% inoculum)                  

                                      TABLE 9                                     __________________________________________________________________________    Mouse Blood Levels of Selected Beta-Lactams After Oral Administration         with BL-P2013 as Mutual Pro-Drugs or Mixtures                                 Test Number 1                                                                              Blood Level (μg/ml)                                                   Dose Minutes After Administration                                                                     AUC                                           Compound                                                                              (mg/kg)                                                                            15  30 60 90 120                                                                              150                                                                              (μg · h/ml)                       __________________________________________________________________________    BL-P2043                                                                              50   5.1 5  3  1.8                                                                              1.3                                                                              1.1                                                                              6.5                                                   38   5.3 4.3                                                                              2  1.1                                                                              0.8                                                                              0.6                                                                              5                                             Ampicillin +                                                                          50   1.8 3.2                                                                              3.8                                                                              3  2.4                                                                              2  6.8                                           BL-P2013                                                                              38   1.1 1.7                                                                              2.2                                                                              1.6                                                                              1.3                                                                              1.1                                                                              3.8                                           BL-P2048                                                                              50   8*  8.2                                                                              6.3                                                                              3.7                                                                              2.6                                                                              1.9                                                                              11.9                                                  38   10  9.3                                                                              4  1.3                                                                              0.9                                                                              0.6                                                                              9.2                                           Hetacillin +                                                                          50   1.2*                                                                              2.1                                                                              3.1                                                                              3.3                                                                              3.1                                                                              2.1                                                                              6.3                                           BL-P2013                                                                              38   1.2 2  2.1                                                                              1.7                                                                              1.4                                                                              0.8                                                                              3.9                                           BL-P2044                                                                              50   1.3 2.2                                                                              2.5                                                                              2.3                                                                              1.9                                                                              2.3                                                                              5.1                                                   38   0.9 1.2                                                                              1.4                                                                              1  0.8                                                                              0.6                                                                              2.4                                           Amoxicillin +                                                                         50   5.5 8.7                                                                              7.4                                                                              5.7                                                                              3.9                                                                              3.1                                                                              13.9                                          BL-P2013                                                                              38   1.4 2.3                                                                              2.8                                                                              2.1                                                                              1.7                                                                              1.4                                                                              4.9                                           BL-P2049                                                                              50   3.7**                                                                             4.7                                                                              4.7                                                                              4.1                                                                              3.7                                                                              3.3                                                                              9.8                                                   38   4.1 4.6                                                                              3.1                                                                              2.2                                                                              1.6                                                                              1.2                                                                              6.5                                           Heta.-Amox. +                                                                         50   3.6**                                                                             7.5                                                                              8.8                                                                              6.6                                                                              5.3                                                                              3.8                                                                              15                                            BL-P2013                                                                              38   1.6 2.6                                                                              2.6                                                                              2.1                                                                              1.8                                                                              1.4                                                                              5                                             Ampicillin                                                                            50   2.2 4  3.3                                                                              2.7                                                                              1.7                                                                              1.2                                                                              6.2                                           Amoxicillin                                                                           50   4.2 7  5  3.6                                                                              2.2                                                                              1.8                                                                              9.5                                           BL-P2013                                                                              38   1.6 2.3                                                                              2.1                                                                              2  1.6                                                                              1.2                                                                              4.4                                           __________________________________________________________________________     *Calculated in terms of ampicillin.                                           **Calculated in terms of amoxicillin.                                         Compounds were solubilized in propylene glycol, except for the                Hetacillin:BLP2013 and Heta. amoxicillin:BLP2013 mixtures (suspensions).      Assay organism: E. coli A9675 for BLP2013; B. subtilis ATCC 6633 for the      others.                                                                       Data based on 2 tests each for the prodrugs, and 2 to 5 tests for the         others.                                                                  

                                      TABLE 10                                    __________________________________________________________________________    Mouse Blood Levels of Selected Beta-Lactams After Oral Administration         with                                                                          BL-P2013 as Mutual Pro-Drugs or Mixtures                                      Test Number 2                                                                                 Blood Level (μg/ml)                                        Dose       Dosage                                                                             15 30 60 90 120                                                                              150 AUC                                         Compound                                                                           (mg/kg)                                                                            Form.sup.a                                                                         Minutes After Administration                                                                     (μg.h/ml)                               __________________________________________________________________________    BL-P2048                                                                            50   PPG  8.sup.b                                                                          8.2                                                                              6.3                                                                              3.7                                                                              2.6                                                                              1.9 11.9                                             50   TCW↓                                                                        5.4.sup.b                                                                        5.4                                                                              4.5                                                                              3.1                                                                              2.4                                                                              1.9 8.9                                              38   PPG  10 9.3                                                                              4  1.3                                                                              0.9                                                                              0.6 9.2                                              38   TCW↓                                                                        8.6                                                                              6.9                                                                              4.2                                                                              1.5                                                                              0.9                                                                              0.6 8.2                                        Hetacillin                                                                          50   PPG↓                                                                        1.2.sup.b                                                                        2.1                                                                              3  3.3                                                                              3.1                                                                              2.1 6.3                                        +     50   TCW  1.2.sup.b                                                                        2.3                                                                              2.6                                                                              2.2                                                                              1.8                                                                              1.2 4.8                                        BL-P2013                                                                            38   PPG↓                                                                        1.2                                                                              2  2.1                                                                              1.7                                                                              1.4                                                                              0.8 3.9                                              38   TCW  1.7                                                                              2.2                                                                              1.7                                                                              1.1                                                                              0.9                                                                              0.6 3.3                                        BL-P2049                                                                            50   PPG  3.7.sup.c                                                                        4.7                                                                              4.7                                                                              4.1                                                                              3.7                                                                              3.3 9.8                                              50   TCW↓                                                                        2.9.sup.c                                                                        4.3                                                                              3.8                                                                              3.3                                                                              2.9                                                                              2.2 8.4                                              38   PPG  4.1                                                                              4.6                                                                              3.1                                                                              2.2                                                                              1.6                                                                              1.2 6.5                                              38   TCW↓                                                                        3.1                                                                              2.7                                                                              1.2                                                                              0.8                                                                              0.6                                                                              <0.5                                                                              2.9                                        Heta- 50   PPG↓                                                                        3.6.sup.c                                                                        7.5                                                                              8.8                                                                              6.6                                                                              5.3                                                                              3.8 15                                         Amoxicillin                                                                         50   TCW  3.6.sup.c                                                                        6.4                                                                              7.3                                                                              5  3.4                                                                              2.2 11.7                                       +     38   PPG↓                                                                        1.6                                                                              2.6                                                                              2.6                                                                              2.1                                                                              1.8                                                                              1.4 5                                          BL-P2013                                                                            38   TCW  1.2                                                                              1.6                                                                              1.4                                                                              0.9                                                                              0.6                                                                              <0.5                                                                              2.2                                        Ampicillin                                                                          50   PPG  2.2                                                                              4  3.3                                                                              2.7                                                                              1.7                                                                              1.2 6.2                                              50   TCW  2.2                                                                              3.2                                                                              2.6                                                                              1.7                                                                              0.9                                                                              0.7 3.9                                        Amoxicillin                                                                         50   PPG  4.2                                                                              7  5  3.6                                                                              2.2                                                                              1.8 9.5                                              50   TCW  7.5                                                                              11 9  5.7                                                                              3.1                                                                              1.9 15.4                                       BL-P2013                                                                            38   PPG  1.6                                                                              2.3                                                                              2.1                                                                              2  1.6                                                                              1.2 4.4                                              38   TCW  1.4                                                                              1.8                                                                              1.4                                                                              0.9                                                                              0.6                                                                              0.5 2.4                                        __________________________________________________________________________     .sup.a PPG = Propylene glycol.                                                TCW = Tweencarboxymethylcellulose-water.                                      ↓ = Drug administered as suspension.                                   .sup.b Calculated in terms of ampicillin.                                     .sup.c Calculated in terms of amoxicillin.                                    Assay organism: E. coli A9675 for BLP2013; B. subtilis ATCC 6633 for the      others.                                                                       Data based on two tests each for the prodrugs, and 1 to 3 tests for the       others.                                                                  

                  TABLE 11                                                        ______________________________________                                        Antibacterial Activity of Ceforanide Alone and                                in Combination with BL-P2013                                                              MIC (μg/ml).sup.a                                                                          Ceforanide:                                                 No. of            BL-P2013                                          Organism  Strains Ceforanide                                                                              1:1     BL-P2013                                  ______________________________________                                        S. faecalis                                                                             3       >125      >125:>125                                                                             >125                                      E. aerogenes                                                                            4       3.4       1.4:1.4 >125                                                1       >125      27:27   16                                                  5       >125      27:27   >125                                      E. cloacae                                                                              1       22.6      2:2     >125                                                14      >125      77:77   >125                                                1       >125      >125:>125                                                                             >125                                      Citrobacter sp.                                                                         1       >125      16:16   16                                        C. freundii                                                                             1       >125      63:63   >125                                      P. mirabilis                                                                            1       0.25      0.5:0.5 >125                                                3       25.4      1.6:1.6 >125                                      P. vulgaris                                                                             1       0.5       0.5:0.5 >125                                                9       85        2.6:2.6 >125                                                4       >125      4.8:4.8 >125                                      M. morganii                                                                             8       63        1.8:1.8 >125                                                8       >125      3.1:3.1 >125                                      P. rettgeri                                                                             1       0.06      0.06:0.06                                                                             >125                                                1       16        16:16   >125                                      P. stuartii                                                                             1       0.35      0.5:0.5 125                                                 4       45        27:27   125                                       S. marcescens                                                                           10      125       51:51   125                                                 6       125       125:125 125                                       P. aeruginosa                                                                           16      125       125:125 125                                       ______________________________________                                         .sup.a Geometric mean value where applicable.                            

                                      TABLE 12                                    __________________________________________________________________________    Therapeutic Efficacy of β-Lactam Antibiotics and β-Lactamase        Inhibitors as                                                                 Mutual Pro-Drugs or Mixtures After Oral Administration to Mice                Experimentally Infected with Different Bacterial Strains                               PD.sub.50 /Treatment.sup.a (mg/kg)                                                                  Amoxicillin                                             BL-P                                                                              Hetacillin                                                                           BL-P                                                                              Heta-Amox.                                                                           + BL-P2013                                     Organism 2048.sup.b                                                                        + BL-P2013                                                                           2049.sup.b                                                                        + BL-P2013                                                                           (1:1)                                          __________________________________________________________________________    E. coli                                                                            A-9675                                                                            49  55      28 16     19                                             E. coli                                                                            A21223                                                                            32  21      32 19     21                                             E. coli                                                                            A20649                                                                            89  57      43 25     25                                             S. aureus                                                                          A-9606                                                                            274 121    >374                                                                              121    114                                            S. aureus                                                                          A20379                                                                            400 200    >400                                                                              87     200                                            __________________________________________________________________________     .sup.a Treatment schedule: Mice were infected i.p. with about 5 ×       10.sup.6 organisms of E. coli in 4% mucin and 1 × 10.sup.9 of S.        aureus in 2% mucin. Drugs were administered p.o. at 0 and 2 hour              postchallenge.                                                                .sup.b Mutual ProDrugs: BLP2048 (hetacillin:BLP2013) and BLP2049              (hetaamoxicillin:BL-P2013) as well as their respective mixtures were          administered in a 50:38 ratio. PD.sub.50 values are averages of 2 to 3        tests for E. coli and 1 to 2 tests for S. aureus.                             PD.sub.50 values for the amoxicillin:lactamase inhibitor mixtures are         averages of 1 to 2 tests for E. coli and a single test for S. aureus.    

                                      TABLE 13                                    __________________________________________________________________________    Therapeutic Efficacy of Mutual Pro-Drugs BL-P2043 and BL-P2044 and Their      Components                                                                    After Oral Administration to Mice Experimentally Infected with a Strain       of E. coli                                                                           PD.sub.50 /Treatment.sup.a (mg/kg)                                            BL-P                                                                              Ampicillin                                                                           BL-P                                                                              Amoxicillin        BL-P                                 Organism                                                                             2043.sup.b                                                                        + BL-P2013                                                                           2044.sup.b                                                                        + BL-P2013                                                                           Ampicillin                                                                          Amoxicillin                                                                         2013                                 __________________________________________________________________________    E. coli A9675                                                                        44  57     25  25     >400  >400  >50                                  __________________________________________________________________________     .sup.a Treatment schedule: Mice were infected i.p. with 8 × 10.sup.     organisms in 4% mucin. Drugs were administered p.o. at 0 and 2 hour           postchallenge.                                                                .sup.b Mutual ProDrugs: BLP2043 (ampicillin:BLP2013) and BLP2044              (amoxicillin:BLP2013) as well as their respective mixtures were               administered in a 50:38 ratio. PD.sub.50 values are based on one test.   

The compounds of the present invention are thus useful, given orally andparenterally, for enhancing the effectiveness of β-lactam antibioticsagainst β-lactamase producing bacteria. On a weight basis, the dosage isfrom one-fifth to five times, and preferably equal to, that of theβ-lactam antibiotics. As an example, the compounds of the presentinvention as shown above when used in a 1:1 ratio markedly improved theactivity of ceforanide and amoxicillin against β-lactamase producingstrains of anaerobic Bacteroides such as B. fragilis, B.thetaiotaomicron and other species of that genus and also againstresistant Staphylococcus aureus. The compounds of the present inventionare given either in admixture with or concomitantly with the β-lactamantibiotic with the dosage within the indicated ratio to the known andcustomary dosage of the antibiotic.

Thus, the ability of the compounds of the present invention to enhancethe effectiveness of a β-lactam antibiotic against certainβ-lactamase-producing bacteria makes them valuable for co-administrationwith certain β-lactam antibiotics in the treatment of bacterialinfections in mammals, including man. In the treatment of a bacterialinfection, a compound of the present invention can be comingled with theβ-lactam antibiotic, and the two agents thereby administeredsimultaneously. Alternatively, a compound of the present invention canbe administered as a separate agent during a course of treatment with aβ-lactam antibiotic.

When using a compound of the present invention or salt thereof, toenhance the antibacterial activity of a β-lactam antibiotic, it can beadministered alone, or preferably, in formulation with standardpharmaceutical carriers and diluents. A compound of the presentinvention which is in the acid form or as a pharmaceutically-acceptablesalt thereof, can be administered orally or parenterally; a compound ofthe present invention in the form of an ester which is readilyhydrolyzable in vivo, is best administered orally. Parenteraladministration includes intramuscular, subcutaneous, intraperitoneal andintravenous administration.

When a compound of the present invention is used in the presence of acarrier or diluent, said carrier or diluent is chosen on the basis ofthe intended mode of administration. For example, when considering theoral mode of administration, the compound can be used in the form oftablets, capsules, lozenges, troches, powders, syrups, elixirs, aqueoussolutions and suspensions, and the like, in accordance with standardpharmaceutical practice.

The proportional ratio of active ingredients to carrier will naturallydepend on the chemical nature, solubility, stability and potency of theactive ingredients, as well as the dosage contemplated. However, thesepharmaceutical compositions will likely contain from about 5% to about80% of carrier. In the case of tablets for oral use, carriers which arecommonly used include lactose, sodium citrate and salts of phosphoricacid. Various disintegrants such as starch, and lubricating agents, suchas magnesium stearate, sodium lauryl sulfate and talc, are commonly usedin tablets. For oral administration in capsule form, useful diluents arelactose and high molecular weight polyethylene glycols. When aqueoussuspensions are required for oral use, the active ingredients arecombined with emulsifying and suspending agents. If desired, certainsweetening and/or flavoring agents can be added. For parenteraladministration, which includes intramuscular, intraperitoneal,subcutaneous and intravenous use, sterile solutions of the activeingredients are usually prepared, and the pH or the solutions aresuitably adjusted and buffered. For intravenous use, the totalconcentration of solutes should be controlled to render the preparationisotonic.

Although the prescribing physician will ultimately decide the dosage tobe used in a human subject, the ratio of the daily dosages of a compoundof the present invention, or salt thereof, and the β-lactam antibioticwill normally be in the range from about 1:5 to 5:1, and preferablyabout 1:1. Additionally, the daily oral dosage of each component willnormally be in the range from about 10 to about 200 mg. per kilogram ofbody weight and the daily parenteral dosage of each component willnormally be about 10 to about 100 mg. per kilogram of body weight. Thesefigures are illustrative only, however, and in some cases it may benecessary to use dosages outside these limits.

This invention is capable of industrial application.

I claim:
 1. An ester having the formula ##STR70## wherein R is ##STR71##wherein R¹ is hydrogen or hydroxy and R² is hydrogen, hydroxy, methyl,methoxy or chloro or a nontoxic, pharmaceutically acceptable acidaddition salt of said ester.
 2. An ester having the formula ##STR72##wherein R is ##STR73## wherein R¹ is hydrogen or hydroxy and R² ishydrogen, hydroxy, methyl, methoxy or chloro or a nontoxic,pharmaceutically acceptable acid addition salt of said ester.
 3. Theester having the formula ##STR74##
 4. A nontoxic, pharmaceuticallyacceptable acid addition salt of the ester of claim
 3. 5. Thehydrochloride of the ester of claim
 3. 6. The ester having the formula##STR75##
 7. A nontoxic, pharmaceutically acceptable acid addition saltof the ester of claim
 6. 8. The hydrochloride of the ester of claim 6.